(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
(19) World Intellectual Property Organization
International Bureau
(43) International Publication Date
11 October 2001 (11.10.2001)
PCT
(10) International Publication Number
WO 01/74388 Al
(51) International Patent Classification 7 : A61K 39/395,
A61P 35/00 // C07K 16/24, 16/28, (A61K 39/395, 31:00)
(21) International Application Number: PCT/US01/10382
(22) International Filing Date: 2 April 2001 (02.04.2001)
(25) Filing Language: English
(26) Publication Language: English
(30) Priority Data:
60/193,467 31 March 2000 (31.03.2000) US
(71) Applicant: IDEC PHARMACEUTICALS CORPORA-
TION [US/US]; 11011 Torreyana Road, San Diego, CA
92121 (US).
(72) Inventor: HANNA, Nabil; 14770 Avenida Insurgentes,
ft= Rancho Santa Fe, CA 92067 (US).
^= (74) Agents: TESKIN, Robin, L. et al.; Pillsbury Winthrop
^5 LLP, 1100 New York Avenue, N.W., Washington, DC
5= 20005 (US).
(81) Designated States (national): AE, AG, AL, AM, AT, AU,
AZ, BA, BB, BG, BR, BY, BZ, CA, CH, CN, CO, CR, CU,
CZ, DE, DK, DM, DZ, EE, ES, FI, GB, GD, GE, GH, GM,
HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK,
LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW, MX,
MZ, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL,
TJ, TM, TR, TT, TZ, UA, UG, UZ, VN, YU, ZA, ZW.
(84) Designated States (regional): ARIPO patent (GH, GM,
KE, LS, MW, MZ, SD, SL, SZ, TZ, UG, ZW), Eurasian
patent (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), European
patent (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE,
IT, LU, MC, NL, PT, SE, TR), OAPI patent (BF, BJ, CF,
CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG).
Published:
— with international search report
— before the expiration of the time limit for amending the
claims and to be republished in the event of receipt of
amendments
For two-letter codes and other abbreviations, refer to the "Guid-
ance Notes on Codes and Abbreviations" appearing at the begin-
ning of each regular issue of the PCT Gazette.
qq (54) Title: COMBINED USE OF ANTT-CYTOKINE ANTIBODIES OR ANTAGONISTS AND ANTI-CD20 FOR THE TREAT-
00 MENT OF B CELL LYMPHOMA
|J (57) Abstract: The present invention discloses combined therapies for treating hematologic malignancies, including B cell lym-
***-; phomas and leukemias or solid non-hematologic tumors, comprising administration of anti-cytokine antibodies or antagonists to
^ inhibit the activity of cytokines which play a role in perpetuating the activation of B cells. The administration of such antibodies
and antagonists, particularly anti-ILlO antibodies and antagonists, is particularly useful for avoiding or decreasing the resistance of
Q hematologic malignant cells or solid tumor cells to chemotherapeutic agents and anti-CD20 or anti-CD22 antibodies. The invention
^ also provides combination therapies for solid tumors having B cell involvement comprising the administration of an anti-cytokine
^ antibody and a B cell depleting antibody such as RITUSAN®.
WO 01/74388
PCT/US01/10382
COMBINED USE OF ANTI-CYTOKINE ANTIBODIES
OR ANTAGONISTS AND ANTI-CD20 FOR THE TREATMENT
OF B CELL LYMPHOMA
5 FIELD OF THE INVENTION
The present invention concerns methods for treating hematologic malignancies
including B cell lymphomas and leukemias with anti-cytokine agents such as
antibodies and antagonists, where the targeted cytokines play a potentiating role in the
disease process by stimulating hematologic malignant cells including B lymphoma
10 and leukemia cells. Treatment with anti-cytokine agents in combination with other
known therapies such as chemotherapy and administration of therapeutic antibodies
has been found to provide a synergistic effect.
The invention also embraces the treatment of solid non-hematologic (non-
lymphoid) tumors, e.g., colorectal or liver cancer, which tumors are characterized by
15 B cell involvement, by the administration of a cytokine antibody or cytokine
antagonist, in combination with treatment with an antibody to a B cell target, e.g.
CD20.
BACKGROUND OF THE INVENTION
20 The immune system of vertebrates (for example, primates, which include humans,
apes, monkeys, etc.) consists of a number of organs and cell types which have evolved
to: accurately and specifically recognize foreign microorganisms ("antigen") which
invade the vertebrate-host; specifically bind to such foreign microorganisms; and,
eliminate/destroy such foreign microorganisms. Lymphocytes, as well as other types
25 of cells, are critical to the immune system and to the elimination and destruction of
foreign microorganisms. Lymphocytes are produced in the thymus, spleen and bone
marrow (adult) and represent about 30% of the total white blood cells present in the
circulatory system of humans (adult). There are two major sub-populations of
lymphocytes: T cells and B cells. T cells are responsible for cell mediated immunity,
30 while B cells are responsible for antibody production (humoral immunity). However,
T cells and B cells can be considered interdependent — in a typical immune response,
T cells are activated when the T cell receptor binds to fragments of an antigen that are
bound to major histocompatability complex ("MHC") glycoproteins on the surface of
an antigen presenting cell; such activation causes release of biological mediators
WO 01/74388
PCTAJS01/10382
("interleukins" or "cytokines") which, in essence, stimulate B cells to differentiate and
produce antibody ("immunoglobulins") against the antigen.
Each B cell within the host expresses a different antibody on its surface- thus
one B cell will express antibody specific for one antigen, while another B cell will
5 express antibody specific for a different antigen. Accordingly, B cells are quite
diverse, and this diversity is critical to the immune system. In humans, each B cell
can produce an enormous number of antibody molecules (i.e., about 10 7 to 10 8 ). Such
antibody production most typically ceases (or substantially decreases) when the
foreign antigen has been neutralized. Occasionally, however, proliferation of a
10 particular B cell will continue unabated; such proliferation can result in a cancer
referred to as "B cell lymphoma."
Non-Hodgkin's lymphoma is one type of lymphoma that is characterized by the
malignant growth of B lymphocytes. According to the American Cancer Society, an
estimated 54,000 new cases will be diagnosed, 65% of which will be classified as
15 intermediate- or high-grade lymphoma. Patients diagnosed with intermediate-grade
lymphoma have an average survival rate of two to five years, and patients diagnosed
with high-grade lymphoma survive an average of six months to two years after
diagnosis.
Conventional therapies have included chemotherapy and radiation, possibly
20 accompanied by either autologous or allogeneic bone marrow or stem cell
transplantation if a suitable donor is available, and if the bone marrow contains too
many tumor cells upon harvesting. While patients often respond to conventional
therapies, they usually relapse within several months.
A relatively new approach to treating non-Hodgkin's lymphoma has been to
25 treat patients with a monoclonal antibody directed to a protein on the surface of
cancerous B cells. The antibody may be conjugated to a toxin or radiolabel thereby
affecting cell death after binding. Alternatively, an antibody may be engineered with
human constant regions such that human antibody effector mechanisms are generated
upon antibody binding which result in apoptosis or death of the cell.
WO 01/74388
PCT/US01/10382
Rituximab® (DEC Pharmaceuticals Corporation) is one of a new generation
of monoclonal antibodies developed for the treatment of B cell lymphomas, and in
particular, non-Hodgkin's lymphoma. Rituximab® is a genetically engineered anti-
CD20 monoclonal antibody with murine light-and heavy-chain variable regions and
5 human gamma I heavy-chain and kappa light-chain constant regions. Rituximab® is
more effective than its murine parent in fixing complement and mediating ADCC, and
it mediates CDC in the presence of human complement. The antibody inhibits cell
growth in the B-cell lines FL-18, Ramos, and Raji, sensitizes chemoresistant human
lymphoma cell lines to diphtheria toxin, ricin, CDDP, doxorubicin, and etoposide, and
10 induces apoptosis in the DHL-4 human B-cell lymphoma line in a dose-dependent
manner.
However, many patients are refractory to or relapse following Rituximab®
therapy, as well as chemotherapy. Therefor, there still remains a need for lymphoma
treatments which may be combined with Rituximab® therapy or chemotherapy in
15 order to increase the chance of remission and decrease the rate of relapse in
lymphoma patients.
Many groups have suggested using cytokines for the treatment of various types
of cancers. For instance, Wang et al. suggested that cytokines are "directly cytotoxic
to tumor cells" and showed that interleukin-1 alpha (ILla) potentiated the anti-tumor
20 effect of anti-tumor drugs against several human tumor cells in vitro (Int. J. Cancer
(Nov. 27, 1996) 68(5): 583-587). Bonvida et al. disclose that cytokines have the
potential to "enhance the efficacy of chemotherapeutic agents" and show that
recombinant tumor necrosis factor and the chemotherapeutic agent cisplatin show a
synergistic effect against ovarian cancer cells (Gynecol. Oncol. (Sept. 1990) 38(3):
25 333-339). U.S. Patent No. 5,716,612 teaches that IL-4 may be used to potentiate the
effect of chemotherapeutic agents in the treatment of cancer.
However, some groups have also recognized that cytokines may play a
detrimental role in the development of some cancers. For instance, interleukin-6 (IL6)
has been known for the ability in some instances to inhibit apoptosis of leukemic
30 cells. (See Yonish-Rouach et al. Wild type p53 induces apoptosis of myeloid
WO 01/74388 PCT/US01/10382
leukemic cells and is inhibited by interleukin-6. Nature 352: 345-347 (1991)).
Recently it was shown that JL6 may play a role in the resistance of some leukemic
cells to anti-cancer chemotherapeutic agents, and that, in vitro, anti-IL6 antibody
increases the sensitivity of cisplatin-resistance K562 cells to cisp latin-induced
5 apoptosis. (See Dedoussis et al. Endogenous interleukin 6 conveys resistance to cis-
diamminedichloroplatinum-mediated apoptosis of the K562 human leukemic cell
line).
A potentiating effect on B cells has also been postulated for IL10, the
production of which has been reported to be upregulated in some cell lines derived
10 from B cell lymphomas (See Cortes et al. Interleukin- 10 in non-Hodgkin's
lymphoma. Leuk Lymphoma 26(3-4): 251-259 (July, 1997). However, when the
serum of NHL patients was tested for correlation between IL10 levels and prognosis,
more significance was placed on the levels of viral IL10, which is produced from a
homologous open reading frame BCFR1, located in the genome of the Epstein Barr
15 Virus (EBV). In fact, another group reported at about the same time that IL10 was an
autocrine growth factor for EBV-infected lymphoma cells. (See Beatty et al.
Involvement of EL10 in the autonomous growth of EBV-transformed B cell lines. J.
Immunol. 158(9): 4045-51 (May 1, 1997)). Alternatively, others have hypothesized
that EL6 and IL10 production by macrophages plays a key role in the occurrence of
20 lymphocytic diseases. {See U.S. Patent No. 5,639,600).
It has also been reported that IL10 may work in combination with IL6, IL2 and
TNF-alpha to increase proliferation of non-Hodgkin's lymphoma cells. (See
Voorzanger et al. Interleukin-(IL)10 and IL6 are produced in vivo by non-Hodgkin's
lymphoma cells and act as cooperative growth factors. Cancer Res. 56(23): 5499-505
25 (Dec. 1, 1996). Also statistically significantly higher levels of IL2, IL6, IL8, IL10,
soluble IL2 receptor, soluble transferrin receptor and neopterin were observed in NHL
patients as compared to a control group, although no single parameter was found to be
of prognostic significance. {See Stasi et al. Clinical implications of cytokine and
soluble receptor measurements in patients with newly diagnosed non-Hodgkin's
30 lymphoma. Eur. J. Haemotol. 54(1): 9-17 (Jan., 1995).
-4-
WO 01/74388 PCT/US01/10382
However, there have been j ust as many reports in the literature which have
suggested that cytokines such as IL10 show no correlation to disease progression, and
that such cytokines may actually be helpful in combating lymphoma rather than
contributing to the disease. For instance, when Bonnefoix et al. tested the potential of
5 ten cytokines (JL2, TL3, IL4, TL6, IL10, IL13, G-CSF, GM-CSF, interferon alpha and
interferon gamma) to modulate the spontaneous proliferative response of B-non-
Hodgkin's lymphoma cells of various histological subtypes, this group found that
each cytokine could be either inhibitory or stimulatory depending on the sample, and
that there was no relationship with different histological subtypes, m fact, U.S. Patent
1 0 No. 5,770, 1 90, herein incorporated by reference, suggests administration of IL1 0 in
conjunction with chemotherapeutic agents as a treatment for acute leukemia.
It would be a benefit to lymphoma patients if therapeutic regimens
incorporating anti-cytokine antibodies could be devised whereby such antibodies
could be used to increase the sensitivity of B lymphoma cells to other types of
1 5 therapeutic drugs . It would be particularly helpful if anti-cytokine antibodies could be
administered for the purpose of avoiding or overcoming the resistance of B lymphoma
cells in lymphoma patients to chemotherapeutic agents, and for the purpose of
potentiating the apoptotic activity of .therapeutic antibodies. Such combined treatment
regimens would add to the therapies available to lymphoma patients and potentially
20 decrease the rate of relapse in these patients.
OBJECTS OF THE INVENTION
It is an object of the invention to provide a method of avoiding, decreasing or
overcoming the resistance of hematologic malignant cells or solid non-hematologic
25 tumor cells to at least one chemotherapeutic agent, comprising administering an anti-
cytokine antibody or cytokine antagonist to a patient diagnosed with a hematologic
malignancy prior, concurrent or after administration of at least one chemotherapeutic
agent.
It is a more specific object of the invention to provide a method of avoiding,
30 decreasing or overcoming the resistance of hematologic malignant cells to apoptosis
-5-
WO 01/74388
PCT/US01/10382
induced by a therapeutic agent, comprising administering an anti-cytokine antibody or
cytokine antagonist to a patient diagnosed with a hematologic malignancy.
It is another object of the invention to provide a method of treating a patient
with a hematologic malignancy who has relapsed following chemotherapy,
5 comprising administering an anti-cytokine antibody or cytokine antagonist to said
patient.
It is another object of the invention to provide a method of treating a patient
having a hematologic malignancy who is refractory to chemotherapy, comprising
administering an anti-cytokine antibody or cytokine antagonist to said patient.
10 It is yet another object of the invention to provide a method of treating a
patient with a hematologic malignancy who has relapsed following therapy with a
therapeutic antibody, comprising administering an anti-cytokine antibody or cytokine
antagonist to said patient.
It is still another object of the invention to provide a method of treating a
15 patient with a hematologic malignancy who is refractory to therapy with a therapeutic
antibody, comprising administering an anti-cytokine antibody or cytokine antagonist
to said patient.
It is another object of the invention to provide a method of treating a B cell
lymphoma patient comprising administering to said patient a therapeutically effective
20 amount of an anti-CD20 antibody simultaneously with or consecutively with in either
order an anti-cytokine antibody.
It is another object of the invention to provide a method of treating a solid
non-hematologic (non-lymphoid) tumor wherein B cells elicit a pro-tumor response
by the adniinistration of an anti-cytokine antibody, e.g. an anti-ILlO antibody and at
25 least one B cell depleting antibody, e.g. an anti-CD20 antibody.
It is a more specific object of the invention to provide a method of treating
solid, non-lymphoid tumor involving the digestive system, especially colorectal
cancer or liver cancer by the administration of an anti-cytokine antibody, preferably an
anti-ILlO antibody and a B cell depleting antibody, particularly a depleting anti-CD20
30 antibody.
-6-
WO 01/74388
PCT/US01/10382
SUMMARY OF THE INVENTION
In a first aspect, the present invention relates to the administration of anti-
cytokine antibodies and cytokine antagonists, particularly antibodies to IL10, in
5 combination with chemotherapy drugs and/or therapeutic antibodies to increase the
response rate and response duration in patients with hematological malignancies such
as B cell lymphomas and leukemias or solid non-hematologic tumors, such as breast
cancer, ovarian cancer, testicular cancer and others. Thus, the present invention
relates to methods of treating hematologic malignancies such as B cell lymphomas
1 0 and leukemias by administering to a patient having a hematologic malignancy such as
B cell lymphoma or a leukemia, antibodies directed to B cell receptors and antibodies
or antagonists which interfere with the action of certain cytokines. In particular, the
present invention relates to administration of antibodies to B cell markers which
initiate apoptosis of B lymphoma cells, such as anti-CD20, anti-CD22, anti-CD40,
15 anti-CD23, anti-CD19, anti-CD37 and others identified infra, and antibodies to or
antagonists of cytokines which may interfere with apoptosis, e.g., anti-lLlO.
Combined therapeutic regimens including other treatments which would also benefit
from anti-cytokine therapy, i.e., chemotherapy, are also encompassed. The methods
will find use in particular for treating patients having hematological malignancies
20 such as B lymphomas or leukemias characterized by cells that have become resistant
to chemotherapeutic agents and therapeutic antibodies.
In a second aspect, the present invention provides novel methods of treating
solid non-hematologic (non-lymphoid) tumors having B cell involvement (but not of
B cell origin), particularly cancers wherein B cells elicit a pro-tumor response by the
25 adrninistration of an antibody to a cytokine, e.g., IL10, in conjunction with B cell
specific antibody therapy, particularly a B cell depleting antibody, and preferably
CD20 antibody therapy, optionally in combination with radiotherapy or
chemotherapy. Examples of such solid tumors include colorectal cancer, liver cancer,
breast cancer, lung cancer, prostate cancer, stomach cancer, head and neck cancer,
30 ovarian cancer, testicular cancer, esophageal cancer and others. Suitable
-7-
WO 01/74388 PCT/US01/10382
chemotherapies are discussed infra. These cancers may comprise precancers, Stage I
and H cancers, and advanced cancers, e.g. past Stage II and including solid tumors that
have metastasized.
5 DETAILED DESCRIPTION OF THE INVENTION
In a first aspect, the present invention includes methods of avoiding,
decreasing or overcoming the resistance of hematologic malignant cells including,
e.g., B lymphoma and leukemia cells to at least one chemotherapeutic agent,
comprising administering an anti-cytokine antibody or cytokine antagonist to a patient
1 0 diagnosed with B cell lymphoma.
Often, such resistance by a hematologic malignancy patient's B cells is
mediated by stimulation of the tumorigenic B cells by one or more cytokines such that
the cells fail to respond to apoptotic signals. In such cases, the methods of the present
invention may be described as methods of avoiding, decreasing or overcoming the
1 5 resistance of such tumorigenic B cells to apoptosis, with chemotherapeutic agents
being examples of agents which may induce apoptosis. Also encompassed are
therapeutic antibodies directed to targets on the surface of B cells, such as anti-CD 19,
anti-CD20, anti-CD22, anti-CD40, and anti-CD28 and other B cell targets identified
infra.
20 Because resistance of B cells is often only apparent after a patient has relapsed
following, or is refractory to, a first treatment with a therapeutic agent, the methods of
the present invention will often encompass treating patients with hematologic
malignancies such as B cell lymphoma or leukemia who have relapsed following, or
are refractory to, chemotherapy or therapy with a therapeutic antibody. However, the
25 anti-cytokine antibodies and antagonists of the present invention may also be used in
conjunction with other therapies or prior to other therapies in patients newly
diagnosed with lymphoma to decrease the chance of relapse, and increase the length
and duration of the response to therapy.
The methods of the present invention are appropriate to treat a wide variety of
30 hematologic malignancies, especially B cell lymphomas and leukemias, including but
-8-
WO 01/74388 PCT/US01/10382
not limited to low grade/follicular non-Hodgkin's lymphoma (NHL), small
lymphocytic (SL) NHL, intermediate grade/ follicular NHL, intermediate grade
diffuse NHL, high grade immunoblastic NHL, high grade lymphoblastic NHL, high
grade small non-cleaved cell NHL, bulky disease NHL and Waldenstrom's
5 MacroglobuUnemia, chronic leukocytic leukemia, acute myelogenous leukemia, acute
lymphoblastic leukemia, chronic lymphocytic leukemia, chronic myelogenous
leukemia, lymphoblastic leukemia, lymphocytic leukemia, monocytic leukemia,
myelogenous leukemia, and promyelocytic leukemia. It should be clear to those of
skill in the art that these lymphomas will often have different names due to changing
10 systems of classification, and that patients having lymphomas and leukemias
classified under different names may also benefit from the combined therapeutic
regimens of the present invention.
For instance, a recent classification system proposed by European and
American pathologists is called the Revised European American Lymphoma (REAL)
15 Classification. This classification system recognizes Mantle cell lymphoma and
Marginal cell lymphoma among other peripheral B-cell neoplasms, and separates
some classifications into grades based on cytology, i.e., small cell, mixed small and
large, large cell. It will be understood that all such classified lymphomas may benefit
from the combined therapies of the present invention.
20 The U.S. National Cancer Institute (NCI) has in turn divided some of the
REAL classes into more clinically useful "indolent" or "aggressive" lymphoma
designations. Indolent lymphomas include follicular cell lymphomas, separated into
cytology "grades," diffuse small lymphocytic lymphoma/ chronic lymphocytic
leukemia (CLL), lymphoplasmacytoidV Waldenstrom's Macroglobulinemia, Marginal
25 zone lymphoma and Hairy cell leukemia. Aggressive lymphomas include diffuse
mixed and large cell lymphoma, Burkitt's lymphoma/ diffuse small non-cleaved cell
lymphoma, Lymphoblastic lymphoma, Mantle cell lymphoma and AIDS-related
lymphoma. All that is required is that the extent or duration of response to therapy be
extended as a result of administration of said anti-cytokine antibody or antagonist.
30 But the methods are most preferably used to treat patients having non-Hodgkin's
-9-
WO 01/74388 PCT/US01/10382
lymphoma (NHL), where the present inventors have surprisingly found that
administration of anti-cytokine antibodies and antagonists has a synergistic effect.
Since the effect of cytokines and the identity of detrimental cytokines may vary
among different patients and different types of lymphomas, and the effect of various
5 cytokines on the resistance of B lymphoma cells may vary with different
chemotherapeutic and immunotherapeutic agents, it is suggested that the levels of the
respective cytokines in individual patients be tested before the patients are
administered the anti-cytokine therapy.
In a second aspect, the invention provides a method of treating solid, non-
10 hematologic tumors wherein B cells elicit a protein response (promote tumor growth
and/or metastasis) comprising the admimstration of a anti-cytokine antibody, e.g. an
anti-ILlO antibody, and an antibody to a B cell target, preferably an anti-CD20
antibody having B cell depleting activity. However, the invention includes the usage
of antibodies to other B cell targets identified infra. Also, this aspect further includes
15 the additional use of chemotherapy and/or radiotherapy.
A variety of chemotherapeutic agents have been applied to the treatment of
different types of cancers, and the methods of the present invention will avoid,
decrease or overcome the resistance of malignant, e.g. lymphoma, cells to at least one,
but possibly several, of these chemotherapeutic agents. In particular, chemotherapies
20 which may benefit by supplemental anti-cytokine therapy include but are not limited
to CHOP, ICE, Mitozantrone, Cytarabine, DVP, ATRA, Idarubicin, hoelzer
chemotherapy regime, La La chemotherapy regime, ABVD, CEOP, 2-CdA, FLAG &
IDA with or without subsequent G-CSF treatment), VAD, M & P, C-Weekly, ABCM,
MOPP, DHAP, methotrexate, doxorubicin, daunorubicin, tamoxifen, toremifene, and
25 cisplatin. Other chemotherapeutic agents are identified infra in the section relating to
preferred embodiments.
There are likely to be a variety of cytokines which play a detrimental,
stimulatory role in hematologic or non-hematologic malignancies including leukemic
and lymphoma diseases, either alone or in cooperation with other cytokines. Thus,
30 depending on the patient and the disease, more than one anti-cytokine antibody or
-10-
WO 01/74388
PCT/US01/10382
antagonist may benefit a particular patient as a supplemental therapy. Those
cytokines include but are not limited to IL2, IL6, IL10 and TNF-alpha. Other
appropriate cytokines are identified infra in the preferred embodiments. For non-
Hodgkin's lymphoma, the preferred anti-cytokine treatment will comprise anti-ILlO
5 therapy.
There are several anti-ILlO antibodies which are known in the art and may be
used for the purposes of the present invention. U.S. Patent No. 5,871,725 describes a
rat anti-human antibody designated 19F1. Another anti-ILlO antibody, alpha-ILlO, is
described in U.S. Patent No. 5,837,293. Anti-ILlO antibodies are also described in
10 Tim R. Mosmann, et al., "Isolation of Monoclonal Antibodies Specific For IL-4, IL-5,
IL-6, and a New Th2-Specific Cytokine (TL-1 0), Cytokine Synthesis Inhibitory Factor,
By Using A Solid Phase Radiohmmmoadsorbent Assay," The Journal of Immunology,
145(9):2938-2945, Nov. 1, 1990. Antagonists may take the form of proteins which
compete for receptor binding, e.g., which lack the ability to activate the receptor while
15 blocking IL-10 binding, or IL-10 binding molecules, such as antibodies. The term
antibody should be understood as encompassing antibody fragments as well as whole
antibodies, i.e., Fab, Fab 2 and Fv fragments. Antibodies may be isolated by
immunizing another animal with human IL-10, but then may be humanized using
method known in the art to decrease their immunogenicity once they are administered
20 to a human patient.
The appropriate dosage of anti-cytokine antibody will depend on the cytokine
targeted, the results of preliminary serum profiles in individual patients, the type of
lymphoma being treated and the stage of disease. For anti-ILlO antibodies in the
treatment of newly diagnosed low-grade non-Hodgkin's lymphoma, the preferred
25 dosage may range from .001 mg to 100 mg/kg, preferably from about 0.1 to 100
mg/kg, and most typically about 0.4 to 20 mg/kg body weight, depending on whether
the antibody is administered concurrently with or prior to another therapeutic agent.
Preferably, the anti-cytokine antibody is administered concurrent or prior to a
chemotherapeutic agent or other therapy, typically from about one hour prior, to about
- 11-
WO 01/74388
PCIYUS01/10382
one month prior, preferably within one to seven days prior to administration of
chemotherapeutic or other agent.
Also included in the present invention are kits for accomphshing the disclosed
methods. A kit according to the present invention comprises at least one anti-cytokine
5 antibody or antagonist which may be readily admixed or resuspended with a
pharmaceutically acceptable carrier and conveniently injected into a lymphoma
patient. In cases where the serum of a lymphoma patient is preferably tested for
cytokine profiles prior to administration of said anti-cytokine antibody or antagonist,
the kit may also or alternatively comprise reagents and materials for testing the
10 relative amounts of various cytokines in the patient's serum.
Also encompassed in the present invention are combined therapeutic methods
of treating hematologic malignancies such as B cell lymphoma and leukemias
comprising administering to a patient with a hematologic malignancy a therapeutically
effective amount of a therapeutic antibody simultaneously with or consecutively with
15 in either order an anti-cytokine antibody. Therapeutic antibodies are defined as those
which bind to receptors on the surface of hematologic malignant cells, e.g.,
tumorigenic B cells, and mediate their destruction or depletion when they bind, i.e.,
anti-CD20, anti-CD19, anti-CD22, anti-CD21, anti-CD23, anti-CD37, and other B
cell targets identified infra. While the anti-cytokine agents of the present invention
20 will have some beneficial effect alone in that they block cytokine-mediated
proliferation of tumorigenic B cells, the combined administration of the therapeutic
antibodies with the anti-cytokine agents will have a synergistic effect in that the
duration and/or extent of response will be better than the additive effect of both types
of therapies applied independently.
25 While not wishing to be held to the following theory, the present inventors
believe that the synergistic effects seen by co-administering the anti-cytokine agents
of the present invention are related to the inhibition of the targeted cytokine which
may usually have the effect to inhibit apoptosis. Accordingly, when the anti-cytokine
agents of the present invention are combined with an agent which acts by inducing
30 apoptosis, e.g., anti-CD20, anti-CD22, anti-CD19, anti-CD21, anti-CD23, or anti-
-12-
WO 01/74388
PCT/US01/10382
CD40 antibodies, the combined administration shows a synergetic effect well beyond
the additive effect of either agent alone.
Never-the-less, this does not preclude the use of the anti-cytokine antibodies
and antagonists of the present invention in combined therapies with other antibodies
5 or therapeutic agents whose efficacy is not facilitated via apoptosis. For instance,
radiolabeled antibodies facilitate the destruction of tumor cells by binding to the B
cell surface and delivering a lethal dose of radiation. Such antibodies, as well as
antibodies conjugated to toxins, may also be used in conjunction with the anti-
cytokine agents of the present invention. Preferred radiolabeled antibodies are those
1 0 labeled with yttrium-[90] C°Y). A particularly preferred radiolabeled antibody is
Zevelin (IDEC Pharmaceuticals Corporation), which is an anti-CD20 antibody
conjugated to 90 Y.
The combined therapeutic methods of the present invention may further
comprise administration of at least one chemotherapeutic agent or regimen, where
1 5 such chemotherapy includes, by way of example, CHOP, ICE, Mitozantrone,
Cytarabine, DVP, ATRA, Idarubicin, hoelzer chemotherapy regime, La La
chemotherapy regime, ABVD, CEOP, 2-CdA, FLAG & IDA with or without
subsequent G-CSF treatment), VAD, M & P, C-Weekly, ABCM, MOPP, DHAP,
doxorubicin, cisplatin, daunorubicin, tamoxifen, toremifene, and methotrexate as well
20 as the additional chemotherapeutic agents identified infra. A preferred
chemotherapeutic regimen for the treatment of non-Hodgkin's lymphoma patients is
CHOP. The anti-cytokine antibody or antagonist is preferably administered prior to
the B cell target antibody, e.g., anti-CD20, CD22, CD 19 or CD40, and/or
chemotherapy, such that proliferation of B lymphoma cells as a result of the targeted
25 cytokine is quelled prior to administration of the B cell therapeutic. As described
above, the target cytokine may be IL2, IL6, IL10 or TNF-alpha among others,
depending on the patient's cytokine profile prior to treatment, but preferably the
targeted cytokine is IL10.
As mentioned above, therapeutic antibodies of the present invention may be
30 any antibody which targets a molecule expressed on the surface of B cells, particularly
- 13-
WO 01/74388 PCT/US01/10382
one having B cell depleting activity. A listing of suitable B cell targets is identified
infra.
Depending on the patient and extent of disease, the anti-B cell target binding
antibody, e.g., Rituximab® may be administered at a dosage ranging from .01 to about
5 100 mg/kg, more preferably from about .1 to 50 mg/kg, and most preferably from
about .4 to 20 mg/kg of body weight. Effective dosages may be lower in combined
therapeutic regimens which include anti-cytokine agents, because the proliferative
potential of B lymphoma cells will be reduced. Again, effective doses will depend on
the chosen anti-cytokine therapy, and the relative levels of potentiating cytokine in the
10 patient's serum.
The combined therapies of the present invention are also suitable for treating a
wide range of lymphomas, including but not limited to low grade/follicular non-
Hodgkin's lymphoma (NHL), small lymphocytic (SL) NHL, intermediate grade/
follicular NHL, intermediate grade diffuse NHL, high grade immunoblastic NHL,
1 5 high grade lymphoblastic NHL, high grade small non-cleaved cell NHL, bulky disease
NHL and Waldenstrom's Macroglobulinemia, chronic leukocytic leukemia, acute
myelogenous leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia,
chronic myelogenous leukemia, lymphoblastic leukemia, lymphocytic leukemia,
monocytic leukemia, myelogenous leukemia, and promyelocytic leukemia Preferred
20 targeted diseases are non-Hodgkin's lymphoma (NHL), and particularly low-grade,
follicular NHL. Again, it may be helpful for the serum of the lymphoma patient to be
tested for cytokine profiles prior to administration of the anti-cytokine antibody or
antagonist.
As already discussed, the combination therapies provided herein, particularly
25 the combined usage of an anti-cytokine antibody, e.g. anti-ILlO and an anti-B cell
target antibody, e.g. anti-CD20, are also useful for treating solid, non-hematologic
(non-lymphoid) cancers, including by way of example, colorectal cancer, liver cancer,
and other digestive cancers, breast cancer, esophageal cancer, head and neck cancer,
lung cancer, ovarian cancer, prostate cancer and testicular cancer. These cancers my
30 be in early, intermediate or advanced stages, e.g. metastasis.
-14-
WO 01/74388
PCT/US01/10382
The present invention also encompasses kits for administering the therapeutic
antibody and the anti-cytokine antibody or antagonist according to the disclosed
methods. Kits may comprise more than one type of therapeutic antibody and more
than one anti-cytokine agent. Kits may also comprise reagents and materials for
5 testing cytokine profile prior to administration of the therapeutic antibody and anti-
cytokine antibody or antagonist.
As noted, the invention further embraces the treatment of solid, non-lymphoid
tumors by the administration of an anti-cytokine antibody, e.g., an anti-ILlO antibody,
and a B cell specific antibody, preferably an antibody having substantial B cell
1 0 depleting activity such as RITUXAN® . It ahs been reported that some solid tumors
apparently have B cell involvement. That is to say that the B cells are somehow
involved in promoting or mamtaining the tumorigenic state and may impede the
body's immune defense system against such tumor. With respect thereto, WO 020864
Al, incorporated by reference herein, which identifies Biocrystal Inc. as the Applicant
1 5 describes the treatment of solid, non-lymphoid tumor using antibodies that target B
cells, including Rituxan®. It was reported therein that this treatment resulted in
pronounced anti-tumor responses, even in patients with advanced colorectal cancer,
lung cancer and liver cancer.
By contrast, the present invention provides an improved combination therapy,
20 wherein solid, non-lymphoid tumors are treated by use of an anti-cytokine antibody,
such as anti-ILlO and a B cell depleting antibody, such as an anti-CD20 antibody.
This combination regimen should afford an enhanced method of treating solid
tumors, particularly those wherein B cells are involved, but are not themselves the
cancerous cells. In this regimen, the cytokine antagonist, e.g., anti-cytokine antibody
25 and the B cell depleting antibody, e.g., Rituxan® will be administered separately or
together and in either order.
Additionally, this regimen may include the use of radiotherapy, e.g., external
beam irradiation, total body irradiation, ra<noimmunotherapy or chemotherapy.
Suitable chemotherapies are identified infra. The radioimmunotherapy may comprise
-15-
WO 01/74388 PCT/US01/10382
treatment with a radiolabeled antibody that binds a target expressed by the solid
tumor.
Typically, the anti-cytokine antibody will be administered prior to the B cell
depleting antibody. It is anticipated that this combination therapy will be suitable for
5 treating any solid tumor having B cell involvement. Suitable examples of solid
tumors have been identified previously. One noteworthy example is colorectal cancer.
In this embodiment, the B cell depleting antibody and cytokine will be
administered such that it s delivered to the solid tumor site. Preferably, the antibodies
will be injected proximate or directly at the tumor site, e.g., by intravenous injection at
10 a vein proximate to the tumor.
This combination regimen cell results in remission or shrinkage of the solid
tumor, e.g., a lung or colorectal tumor.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
15 In order to further describe the preferred embodiments and full scope of the
invention, the following definitions are provided.
I. Definitions
"Cytokine antagonist" is a compound that inhibits or blocks the expression
and/or activity of a cytokine, e.g. an interleukin or interferon or another cytokine.
20 A "B cell surface marker" or "B cell target" or "B cell antigen" herein is an
antigen expressed on the surface of a B cell which can be targeted with an antagonist
which binds thereto. Exemplary B cell surface markers include the CD10, CD19,
CD20, CD21, CD22, CD23, CD24, CD37, CD53, CD72, CD73, CD74, CDw75,
CDw76, CD77, CDw78, CD79a, CD79b, CD80, CD81, CD82, CD83, CDw84, CD85
25 and CD86 leukocyte surface markers: The B cell surface marker of particular interest
is preferentially expressed on B cells compared to other non-B cell tissues of a
mammal and may be expressed on both precursor B cells and mature B cells. In one
embodiment, the marker is one, like CD20 or CD 19, which is found on B cells
throughout differentiation of the lineage from the stem cell stage up to a point just
30 prior to terminal differentiation into plasma cells. The preferred B cell surface
- 16-
WO 01/74388
PCTAJS01/10382
markers herein are CD19 and CD20.
The "CD20" antigen is a - 35 kDa, non-glycosylated phosphoprotein found on
the surface of greater than 90% of B cells from peripheral blood or lymphoid organs.
CD20 is expressed during early pre-B cell development and remains until plasma cell
5 differentiation. CD20 is present on both normal B cells as well as malignant B cells.
Other names for CD20 in the literature include "B-lymphocyte-restricted antigen" and
"Bp35". The CD20 antigen is described in Clark et al. PNAS (USA) 82:1766 (1985),
for example. The "CD19" antigen refers to a -90kDa antigen identified, for example,
by the HD237-CD19 or 134 antibody (Kiesel et al. Leukemia Research 11, 12: 1 1 19
10 (1987)). Like CD20, CD19 is found on cells throughout differentiation of the lineage
from the stem cell stage up to a point just prior to terminal differentiation into plasma
cells. Binding of an antagonist to CD 19 may cause internalization of the CD 19
antigen.
A "hematologic malignancy" includes any malignancy associated with cells in
15 the bloodstream. Examples thereof include B and T cell lymphomas, leukemias
including but not limited to low grade/follicular non-Hodgkin's lymphoma (NHL),
small lymphocytic (SL) NHL, intermediate grade/ follicular NHL, intermediate grade
diffuse NHL, high grade immunoblastic NHL, high grade lymphoblastic NHL, high
grade small non-cleaved cell NHL, bulky disease NHL and Waldenstrom's
20 Macroglobulinemia, chronic leukocytic leukemia, acute myelogenous leukemia, acute
lymphoblastic leukemia, chronic lymphocytic leukemia, chronic myelogenous
leukemia, lymphoblastic leukemia, lymphocytic leukemia, monocytic leukemia,
myelogenous leukemia, and promyelocytic leukemia. It should be clear to those of
skill in the art that these lymphomas will often have different names due to changing
25 systems of classification (as previously discussed), and that patients having
lymphomas and leukemias classified under different names may also benefit from the
combined therapeutic regimens of the present invention.
A solid, non-hematologic (non-lymphoid) tumor refers to a non-hematologic
malignancy having B cell involvement, i.e., where B cells are involved in a
30 "protumor" response. Such solid tumors are characterized by palpable tumors,
-17-
WO 01/74388 PCTYUS01/10382
typically at least 0.5 mm in diameter, more typically at least 1 .0 mm in diameter.
Examples thereof include colorectal cancer, liver cancer, breast cancer, lung cancer,
head and neck cancer, stomach cancer, testicular cancer, prostate cancer, ovarian
cancer, uterine cancer and others. These cancers may be in the early stages
5 (precancer), intermediate (Stages I and II) or advanced, including solid tumors that
have metastasized. These solid tumors will preferably be cancers wherein B cells
elicit a protumor response, i.e. the presence of B cells is involved in tumor
development, maintenance or metastasis.
A B cell "antagonist" is a molecule which, upon binding to a B cell surface
10 marker, destroys or depletes B cells in a mammal and/or interferes with one or more B
cell functions, e.g. by reducing or preventing a humoral response elicited by the B
cell. The antagonist preferably is able to deplete B cells (i.e. reduce circulating B cell
levels) in a mammal treated therewith. Such depletion may be achieved via various
mechanisms such antibody-dependent cell mediated cytotoxicity (ADCC) and/or
1 5 complement dependent cytotoxicity (CDC), inhibition of B cell proliferation and/or
induction of B cell death (e.g. via apoptosis). Antagonists included within the scope of
the present invention include antibodies, synthetic or native sequence peptides and
small molecule antagonists which bind to the B cell marker, optionally conjugated
with or fused to a cytotoxic agent. The preferred antagonist comprises an antibody,
20 more preferably a B cell depleting antibody.
"Antibody-dependent cell-mediated cytotoxicity" and "ADCC" refer to a
cell-mediated reaction in which nonspecific cytotoxic cells that express Fc receptors
(FcRs) (e.g. Natural Killer (NK) cells, neutrophils, and macrophages) recognize
bound antibody on a target cell and subsequently cause lysis of the target cell. The
25 primary cells for mediating ADCC, NK cells, express FcyRIH only, whereas
monocytes express FcyRI, FcyRII and FcyRHI. FcR expression on hematopoietic
cells in summarized is Table 3 on page 464 of Ravetch and Kinet, Annu. Rev.
Immunol 9:457-92 (1991). To assess ADCC activity of a molecule of interest, an in
vitro ADCC assay, such as that described in US Patent No. 5,500,362 or 5,821,337
30 may be performed. Useful effector cells for such assays include peripheral blood
- 18-
WO 01/74388
PCT/US01/10382
mononuclear cells (PBMC) and Natural Killer (NK) cells. Alternatively, or
additionally, ADCC activity of the molecule of interest may be assessed in vivo, e.g.,
in a animal model such as that disclosed in Clynes et al. PNAS (USA) 95:652-656
(1998).
5 "Human effector cells" are leukocytes which express one or more FcRs and
perform effector functions. Preferably, the cells express at least FcyRlH and carry out
ADCC effector function. Examples of human leukocytes which mediate ADCC
include peripheral blood mononuclear cells (PBMC), natural killer (NK) cells,
monocytes, cytotoxic T cells and neutrophils; with PBMCs and NK cells being
10 preferred.
The terms "Fc receptor" or "FCR"are used to describe a receptor that binds to
the Fc region of an antibody.
The preferred FcR is a native sequence human FcR. Moreover, a preferred
FcR is one which binds an IgG antibody (a gamma receptor) and includes receptors of
15 the FcyRI, FcyRII, and Fey RIE subclasses, including allelic variants and alternatively
spliced forms of these receptors. FcyRII receptors include FcyRJIA (an "activating
receptor") and FcyREB (an "inhibiting receptor"), which have similar amino acid
sequences that differ primarily in the cytoplasmic domains thereof. Activating
receptor FcyRUA contains an immunoreceptor tyrosine-based activation motif
20 (ITAM) in its cytoplasmic domain. Inhibiting receptor FcyRUB contains an
immunoreceptor tyrosine-based inhibition motif (ITM) in its cytoplasmic domain,
(see Daeron, Annu. Rev. Immunol. 15:203-234 (1997)). FcRs are reviewed in Ravetch
and Kinet, Annu. Rev. Immunol 9:457-92 (1991); Capel et al., hnmunomethods
4:25-34 (1994); and de Haas et al., J. Lab. Clin. Med. 126:330-41 (1995). Other FcRs,
25 including those to be identified in the future, are encompassed by the term "FCR"
herein. The term also includes the neonatal receptor, FcRn, which is responsible for
the transfer of maternal IgGs to the fetus (Guyer et al., J. Immunol. 1 17:587 (1976)
and Kim et al., J. Immunol. 24:249 (1994)).
"Complement dependent cytotoxicity" or "CDC" refer to the ability of a
30 molecule to lyse a target in the presence of complement. The complement activation
- 19-
WO 01/74388 PCT/US01/10382
pathway is initiated by the binding of the first component of the complement system
(Clq) to a molecule (e.g. an antibody) complexed with a cognate antigen. To assess
complement activation, a CDC assay, e.g. as described in Gazzano-Santoro et al., J.
Immunol. Methods 202: 1 63 (1 996), may be performed.
5 "Growth inhibitory 1 ' antagonists are those which prevent or reduce
proliferation of a cell expressing an antigen to which the antagonist binds. For
example, the antagonist may prevent or reduce proliferation of B cells in vitro and/or
in vivo.
Antagonists which "induce apoptosis" are those which induce programmed
1 0 cell death, e.g. of a B cell, as determined by standard apoptosis assays, such as binding
of annexin V, fragmentation of DNA, cell shrinkage, dilation of endoplasmic
reticulum, cell fragmentation, and/or formation of membrane vesicles (called
apoptotic bodies).
The term "antibody" herein is used in the broadest sense and specifically
15 covers intact monoclonal antibodies, polyclonal antibodies, multispecific antibodies
(e.g. bispecific antibodies) formed from at least two intact antibodies, and antibody
fragments so long as they exhibit the desired biological activity.
"Antibody fragments" comprise a portion of an intact antibody, preferably
comprising the antigen-binding or variable region thereof. Examples of antibody
20 fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies;
single-chain antibody molecules; and multispecific antibodies formed from antibody
fragments.
"Native antibodies" are usually heterotetrameric glycoproteins of about
150,000 daltons, composed of two identical light (L) chains and two identical heavy
25 (H) chains. Each light chain is linked to a heavy chain by one covalent disulfide bond,
while the number of disulfide linkages varies among the heavy chains of different
immunoglobulin isotypes. Each heavy and light chain also has regularly spaced
intrachain disulfide bridges. Each heavy chain has at one end a variable domain (VH)
followed by a number of constant domains. Each light chain has a variable domain at
30 one end (VL) and a constant domain at its other end; the constant domain of the light
-20-
WO 01/74388
PCT/US01/10382
chain is aligned with the first constant domain of the heavy chain, and the light-chain
variable domain is aligned with the variable domain of the heavy chain. Particular
amino acid residues are believed to form an interface between the light chain and
heavy chain variable domains.
5 The term "variable" refers to the fact that certain portions of the variable
domains differ extensively in sequence among antibodies and are used in the binding
and specificity of each particular antibody for its particular antigen. However, the
variability is not evenly distributed throughout the variable domains of antibodies. It
is concentrated in three segments called hypervariable regions both in the light chain
10 and the heavy chain variable domains. The more highly conserved portions of variable
domains are called the framework regions (FRs). The variable domains of native
heavy and light chains each comprise four FRs, largely adopting a P-sheet
configuration, connected by three hypervariable regions, which form loops
connecting, and in some cases forming part of, the (3 sheet structure. The
1 5 hypervariable regions in each chain are held together in close proximity by the FRs
and, with the hypervariable regions from the other chain, contribute to the formation
of the antigen-binding site of antibodies (see Kabat et at, Sequences of Proteins of
Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health,
Bethesda, MD. (1991)). The constant domains are not involved directly in binding an
20 antibody to an antigen, but exhibit various effector functions, such as participation of
the antibody in antibody dependent cellular cytotoxicity (ADCC).
Papain digestion of antibodies produces two identical antigen-binding
fragments, called "Fob" fragments, each with a single antigen-binding site, and a
residual "Fc" fragment, whose name reflects its ability to crystallize readily. Pepsin
25 treatment yields an F(ab'2 fragment that has two antigen-binding sites and is still
capable of crosslinking antigen.
"Fv" is the minimum antibody fragment which contains a complete
antigen-recognition and antigen-binding site. This region consists of a dimer of one
heavy chain and one light chain variable domain in tight, non-covalent association. It
30 is in this configuration that the three hypervariable regions of each variable domain
-21 -
WO 01/74388
PCT/US01/10382
interact to define an antigen-binding site on the surface of the VH-VL dimer.
Collectively, the six hypervariable regions confer antigen binding specificity to the
antibody. However, even a single variable domain (or half of an Fv comprising only
three hypervariable regions specific for an antigen) has the ability to recognize and
5 bind antigen, although at a lower affinity than the entire binding site.
The Fab fragment also contains the constant domain of the light chain and the
first constant domain (CHI) of the heavy chain. Fab' fragments differ from Fab
fragments by the addition of a few residues at the carboxy terminus of the heavy chain
CHI domain including one or more cysteines from the antibody hinge region. Fab'-SH
10 is the designation herein for Fab' in which the cysteine residue(s) of the constant
domains bear at least one free thiol group. F(ab")Z antibody fragments originally were
produced as pairs of Fab' fragments which have hinge cysteines between them. Other
chemical couplings of antibody fragments are also known.
The "light chains" of antibodies (immunoglobulins) from any vertebrate
15 species can be assigned to one of two clearly distinct types, called kappa (x) and
lambda (k), based on the amino acid sequences of their constant domains.
Depending on the amino acid sequence of the constant domain of their heavy
chains, antibodies can be assigned to different classes. There are five major classes of
intact antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these maybe further
20 divided into subclasses (isotypes), e.g., IgG 1, IgG2, IgG3, IgG4, IgA, and IgA2. The
heavy-chain constant domains that correspond to the different classes of antibodies
are called a, 8, s, y, and R, respectively. The subunit structures and three-dimensional
configurations of different classes of immunoglobulins
are well known.
25 "Single-chain Fv" or "scFv" antibody fragments comprise the VH and VL
domains of antibody, wherein these domains are present in a single polypeptide chain.
Preferably, the Fv polypeptide further comprises a polypeptide linker between the VH
and VL domains which enables the scFv to form the desired structure for antigen
binding. For a review of scFv see Pluckthun in The Pharmacology of Monoclonal
30 Antibodies, vol. 1 13, Rosenburg and Moore, eds., Springer- Verlag, New York, pp.
-22-
WO 01/74388
PCT/US01/10382
269-315 (1994).
The term "diabodies" refers to small antibody fragments with two
antigen-binding sites, which fragments comprise a heavy-chain variable domain (VH)
connected to a light-chain variable domain (VL) in the same polypeptide chain (VH -
5 VL). By using a linker that is too short to allow pairing between the two domains on
the same chain, the domains are forced to pair with the complementary domains of
another chain and create two antigen binding sites. Diabodies are described more fully
in, for example, EP 404,097; WO 93/11161; and Hollinger et al, Proc. Nad. Acad.
Sci. USA, 90:6444.-6448 (1993).
10 The term "monoclonal antibody" as used herein refers to an antibody obtained
from a population of substantially homogeneous antibodies, i.e., the individual
antibodies comprising the population are identical except for possible naturally
occurring mutations that may be present in minor amounts. Monoclonal antibodies are
highly specific, being directed against a single antigenic site. Furthermore, in contrast
15 to conventional (polyclonal) antibody preparations which typically include different
antibodies directed against different determinants (epitopes), each monoclonal
antibody is directed against a single determinant on the antigen. In addition to their
specificity, the monoclonal antibodies are advantageous in that they are synthesized
by the hybridoma culture, uncontaminated by other immunoglobulins. The modifier
20 "monoclonal" indicates the character of the antibody as being obtained from a
substantially homogeneous population of antibodies, and is not to be construed as
requiring production of the antibody by any particular method. For example, the
monoclonal antibodies to be used in accordance with the present invention may be
made by the hybridoma method first described by Kohler et al, Nature, 256:495
25 (1975), or may be made by recombinant DNA methods (see, e.g., U.S. Patent No.
4,816,567). The "monoclonal antibodies" may also be isolated from phage antibody
libraries using the techniques described in Clackson et al, Nature, 352:624-628
(1991) and Marks et al, J. Mol. Biol, 222:581-597 (1991), for example.
The monoclonal antibodies herein specifically include "chimeric" antibodies
30 (immunoglobulins) in which a portion of the heavy and/or light chain is identical with
-23-
WO 01/74388
PCT/US01/10382
or homologous to corresponding sequences in antibodies derived from a particular
species or belonging to a particular antibody class or subclass, while the remainder of
the chain(s) is identical with or homologous to corresponding sequences in antibodies
derived from another species or belonging to another antibody class or subclass, as
5 well as fragments of such antibodies, so long as they exhibit the desired biological
activity (U.S. Patent No. 4,816,567; Morrison et al, Proc. Natl. Acad. Sci. USA,
81:6851-6855 (1984)). Chimeric antibodies of interest herein include "primatized"
antibodies comprising variable domain antigen binding sequences derived from a
non-human primate (e.g. Old World Monkey, such as baboon, rhesus or cynomolgus
1 0 monkey) and human constant region sequences (US Pat No. 5,693,780).
"Humanized" forms of non-human (e.g., murine) antibodies are chimeric
antibodies that contain minimal sequence derived from non-human immunoglobulin.
For the most part, humanized antibodies are human immunoglobulins (recipient
antibody) in which residues from a hypervariable region of the recipient are replaced
1 5 by residues from a hypervariable region of a non-human species (donor antibody)
such as mouse, rat, rabbit or nonhuman primate having the desired specificity,
affinity, and capacity. In some instances, framework region (FR) residues of the
human immunoglobulin are replaced by corresponding non-human residues.
Furthermore, humanized antibodies may comprise residues that are not found in the
20 recipient antibody or in the donor antibody. These modifications are made to further
refine antibody performance. In general, the humanized antibody will comprise
substantially all of at least one, and typically two, variable domains, in which all or
substantially all of the hypervariable loops correspond to those of a non-human
immunoglobuhn and all or substantially all of the FRs are those of a human
25 immunoglobulin sequence. The humanized antibody optionally also will comprise at
least a portion of an immunoglobulin constant region (Fc), typically that of a human
immunoglobulin. For further details, see Jones et al. Nature 321:522-525 (1986);
Riechmann et al, Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol.
2:593-596 (1992).
30 The term "hypervariable region" when used herein refers to the amino acid
-24-
WO 01/74388 PCT/US01/10382
residues of an antibody which are responsible for antigen-binding. The hypervariable
region comprises amino acid residues from a "complementarity determining region"
or "CDR" (e.g. residues 24-34 (LI), 50-56 (L2) and 89-97 (L3) in the light chain
variable domain and 3 1-35 (HI), 50-65 (H2) and 95-102 (H3) in the heavy chain
5 variable domain; Kabat et al, Sequences of Proteins of Immunological Interest, 5th
Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991))
and/or those residues from a "hypervariable loop" (e.g. residues 26-32 (LI), 50-52
(L2) and 91-96 (L3) in the light chain variable domain and 26-32 (HI), 53-55 (H2)
and 96-101 (H3) in the heavy chain variable domain; Chothia and Lesk J. Mol. Biol.
10 196:901-917 (1987)). "Framework" or "FR" residues are those variable domain
residues other than the hypervariable region residues as herein defmed. An antagonist
"which binds" an antigen of interest, e.g. a B cell surface marker, is one capable of
binding that antigen with sufficient affinity and/or avidity such that the antagonist is
useful as a therapeutic agent for targeting a cell expressing the antigen.
15 Examples of antibodies which bind the CD20 antigen include: "C2B8" which
is now called "rituximab" ("RITUXAN®") (US Patent No. 5,736,137, expressly
incorporated herein by reference); the yttrium- [90] -labeled 2138 murine antibody
designated "Y2B8" (US Patent No. 5,736,137, expressly incorporated herein by
reference); murine IgG2a "131" optionally labeled with 1311 to generate the
20 "131I-B1" antibody (BEXXARTM) (US Patent No. 5,595,721, expressly incorporated
herein by reference); murine monoclonal antibody " 1F5" (Press et al. Blood
69(2):584-591 (1987)); "chimeric 2H7" antibody (US Patent No. 5,677,180, expressly
incorporated herein by reference); and monoclonal antibodies L27, G28-2, 93-1 133,
B-Cl or NU-B2 available from the International Leukocyte Typing Workshop
25 (V alentine et al, In: Leukocyte Typing III (McMichael, Ed., p. 440, Oxford University
Press (1987)). Examples of antibodies which bind the CD 19 antigen include the
anti-CD 19 antibodies in Hekman et al, Cancer Immunol. Immunother. 32:364-372
(1991) and Vlasveld et al. Cancer Immunol. Immunother. 40:37-47(1995); and the B4
antibody in Kiesel et al. Leukemia Research 11, 12: 1119 (1987).
30 The terms "rituximab" or "RITUXAN®" herein refer to the genetically
-25-
WO 01/74388 PCT/US01/10382
engineered chimeric murine/humati monoclonal antibody directed against the CD20
antigen and designated "C2B8" in US Patent No. 5,736,137, expressly incorporated
herein by reference. The antibody is an IgG, kappa irnmunoglobulin containing
murine light and heavy chain variable region sequences and human constant region
5 sequences. Rituximab has a binding affinity for the CD20 antigen of approximately
8.0nM.
An "isolated" antagonist is one which has been identified and separated and/or
recovered from a component of its natural environment. Contaminant components of
its natural environment are materials which would interfere with diagnostic or
10 therapeutic uses for the antagonist, and may include enzymes, hormones, and other
proteinaceous or nonproteinaceous solutes. In preferred embodiments, the antagonist
will be purified (1) to greater than 95% by weight of antagonist as determined by the
Lowry method, and most preferably more than 99% by weight, (2) to a degree
sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence
15 by use of a spinning cup sequenator, or (3) to homogeneity by SDS-PAGE under
reducing or nonreducing conditions using Coomassie blue or, preferably, silver stain.
Isolated antagonist includes the antagonist in situ within recombinant cells since at
least one component of the antagonist's natural environment will not be present.
Ordinarily, however, isolated antagonist will be prepared by at least one purification
20 step. "Mammal" for purposes of treatment refers to any animal classified as a
mammal, including humans, domestic and farm animals, and zoo, sports, or pet
animals, such as dogs, horses, cats, cows, etc. Preferably, the mammal is human.
"Treatment" refers to both therapeutic treatment and prophylactic or
preventative measures. Those in need of treatment include those already with the
25 disease or disorder as well as those in which the disease or disorder is to be prevented.
Hence, the mammal may have been diagnosed as having the disease or disorder or
may be predisposed or susceptible to the disease.
The expression "therapeutically effective amount" refers to an amount of the
antagonist which is effective for preventing, ameliorating or treating the autoimmune
30 disease in question. The term "immunosuppressive agent" as used herein for adjunct
-26-
WO 01/74388 PCT/US01/10382
therapy refers to substances that act to suppress or mask the immune system of the
mammal being treated herein. This would include substances that suppress cytokine
production, downregulate or suppress self-antigen expression, or mask the MHC
antigens.
5 Examples of such agents include 2-amino-6-aryl-5-substituted pyrirnidines
(see U.S. Pat. No. 4,665,077, the disclosure of which is incorporated herein by
reference); azathioprine; cyclophosphamide; bromocryptine; danazol; dapsone;
glutaraldehyde (which masks the MHC antigens, as described in U.S. Pat. No.
4,120,649); anti-idiotypic antibodies for MHC antigens and MHC fragments;
10 cyclosporin A; steroids such as glucocorticosteroids, e.g., prednisone,
methylprednisolone, and dexamethasone; cytokine or cytokine receptor antagonists
including anti-interferon-y, -(3, or-a antibodies, anti-tumornecrosis factor-a
antibodies, anti-tumornecrosis factor-(i antibodies, anti-mterleukin-2 antibodies and
anti-IL-2 receptor antibodies; anti-LFA-1 antibodies, including anti-CD 11a and anti-
15 CD 18 antibodies; anti-L3T4 antibodies; heterologous anti-lymphocyte globulin; pan-T
antibodies, preferably antiCD3 or anti-CD4/CD4a antibodies; soluble peptide
containing a LFA-3 binding domain (WO 90/08187 published 7/26/90); streptokinase;
TGF-0; streptodornase; RNA or DNA from the host; FK506; RS-61443;
deoxyspergualin; rapamycin; T-cell receptor (Cohen et al, U.S. Pat. No. 5,114,721);
20 T-cell receptor fragments (Offher et al, Science 251 : 430-432 (1991); WO 90/1 1294;
Ianeway, Nature, 341: 482 (1989); and WO 91/01133); and T cell receptor antibodies
(EP 340,109) such as TLOB9.
The term "cytotoxic agent" as used herein refers to a substance that inhibits or
prevents the function of cells and/or causes destruction of cells. The term is intended
25 to include radioactive isotopes (e.g. I 131 , Y 90 , Ar 211 , P 32 , Re 188 , Re 186 , Sm 153 , B 212 and
others), chemotherapeutic agents, and toxins such as small molecule toxins or
enzymatically active toxins of bacterial, fungal, plant or animal origin, or fragments
thereof.
A "chemotherapeutic agent" is a chemical compound useful in the treatment of
30 cancer. Examples of chemotherapeutic agents include alkylating agents such as
-27-
WO 01/74388 PCT/US01/10382
thiotepa and cyclosphosphamide (CYTOXANTM); alkyl sulfonates such as busulfan,
improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa,
and uredopa; ethylenimines and methylamelamines including altretamine,
triethylenemelamine, trietylenephosphoramide, triethylenethiophosphaoramide and
5 trimethylolomelamine; nitrogen mustards such as chlorambucil, chlornaphazine,
cholophosphamide, estramustine, ifosfamide, mechlorethamine, mecMorethamine
oxide hydrochloride, melphalan, novembiehin, phenesterine, prednimustine,
trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin,
fotemustine, lomustine, nimustine, ranimustine; antibiotics such as aclacinomysins,
10 actinomycin, authramycin, azaserine, bleomycins, cactinomycin, calicheamicin,
carabicin, carminomycin, carzinophilin, chromoinycins, dactinomycin, daunorubicin,
detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin,
idambicin, marcellomycin, mitomycins, mycophenolic acid, nogalamycin,
olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin,
15 streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin;
anti-metabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues
such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as
fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as
ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine,
20 doxifluridine, enocitabine, floxuridine, 5-FU; androgens such as calusterone,
dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals
such as aminoglutethimide, mitotane, trilostane; fohc acid replenisher such as frolinic
acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; amsacrine;
bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone;
25 elformthine; elliptinium acetate; etoglucid; gallium nitrate; hydroxyurea; lentinan;
lonidamine; mitoguazone; mitoxantrone; mopidamol; nitracrine; pentostatin;
phenamet; pirarubicin; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK®;
razoxane; sizofrran; spirogermanium; tenuazonic acid; triaziquone; 2,
2\2"-tricMorotriemylamine; urethan; vindesine; dacarbazine; mannomustine;
30 mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C");
-28-
WO 01/74388
PCT/US01/10382
cyclophosphamide; thiotepa; taxoids, e.g. pachtaxel (TAXOLO, Bristol-Myers Squibb
Oncology, Princeton, NJ) and doxetaxel (TAXOTEW, Rh6ne-Poulenc Rorer, Antony,
France); chlorambucil; gemcitabine; 6-tbioguanine; mercaptopurine; methotrexate;
platinum analogs such as cisplatin and carboplatin; vinblastine; platinum; etoposide
5 (VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine;
novantrone; teniposide; daunomycin; aminopterin; xeloda; ibandronate; CPT-11;
topoisomerase inhibitor RFS 2000; difluoromemylorrdthine (DMFO); retinoic acid;
esperamicins; capecitabine; and pharmaceutically acceptable salts, acids or derivatives
of any of the above. Also included in this definition are anti-hormonal agents that act
10 to regulate or inhibit hormone action on tumors such as anti-estrogens including for
example tamoxifen, raloxifene, aromatase inhibiting 4(5)-imidazoles, 4
hydroxytamoxifen, trioxifene, keoxifene, LY 117018, onapristone, and toremifene
(Fareston); and anti-androgens such as flutamide, nilutamide, bicalutamide,
leuprohde, and goserelin; and pharmaceutically acceptable salts, acids or derivatives
15 of any of the above.
The term "cytokine" is a generic term for proteins released by one cell
population which act on another cell as intercellular mediators. Examples of such
cytokines are lymphokines,, monokines, and traditional polypeptide hormones.
Included among the cytokines are growth hormone such as human growth hormone,
20 N-methionyl human growth hormone, and bovine growth hormone; parathyroid
hormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones
such as follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), and
luteinizing hormone (LH); hepatic growth factor; fibroblast growth factor; prolactin;
placental lactogen; tumor necrosis factor-a and -0; mullerian-inhibiting substance;
25 mouse gonadotropin-associated peptide; inhibin; activin; vascular endothelial growth
factor; integrin; thrombopoietin (TPO); nerve growth factors such as NGF-P; platelet
growth factor; transforming growth factors (TGFs) such as TGF-a and TGF-0;
insulin-like growth factor-I and -H; erythropoietin (EPO); osteoinductive factors;
interferons such as interferon-a, -P, and -y; colony stimulating factors (CSFs) such as
30 macrophage-CSF (M-CSF); granulocyte-macrophage-CSF (GM-CSF); and
-29-
WO 01/74388 PCT/US01/10382
granulocyte-CSF (GCSF); interleukins QLs) such as IL-1 , IL-la, IL-2, IL-3, IL-4, IL-5,
JL-6, JL-7, JL-8, JL-9, IL-1 1, IL-12, EL-15; a tumor necrosis factor such as TNF-a or
TNF-P; and other polypeptide factors including LIF and kit ligand (KL). As used
herein, the term cytokine includes proteins from natural sources or from recombinant
5 cell culture and biologically active equivalents of the native sequence cytokines.
The term "prodrug" as used in this application refers to a precursor or
derivative form of a pharmaceutically active substance that is less cytotoxic to tumor
cells compared to the parent drug and is capable of being enzymatically activated or
converted into the more active parent form. See, e.g., Wihnan, "Prodrugs in Cancer
10 Chemotherapy" Biochemical Society Transactions, 14, pp. 375-382, 615th Meeting
Belfast (1986) and Stella et al, "Prodrugs: A Chemical Approach to Targeted Drug
Delivery," Directed Drug Delivery, Borchardt et al, (ed.), pp. 247-267, Humana Press
(1985). The prodrugs of this invention include, but are not limited to,
phosphate-containing prodrugs, thiophosphate-containing prodrugs, sulfate-containing
1 5 prodrugs, peptide-containing prodrugs, D-amino acid-modified prodrugs, glycosylated
prodrugs, (3 -lactam-containing prodrugs, optionally substituted
phenoxyacetamide-containing prodrugs or optionally substituted
phenylacetamide-containing prodrugs, 5 fluorocytosine and other 5-fluorouridine
prodrugs which can be converted into the more active cytotoxic free drag. Examples
20 of cytotoxic drugs that can be derivatized into a prodrug form for use in this invention
include, but are not limited to, those chemotherapeutic agents described above.
A "liposome" is a small vesicle composed of various types of lipids,
phospholipids and/or surfactant which is useful for delivery of a drug (such as the
antagonists disclosed herein and, optionally, a chemotherapeutic agent) to a mammal.
25 The components of the liposome are commonly arranged in a bilayer formation,
similar to the lipid arrangement of biological membranes. The term "package insert"
is used to refer to instructions customarily included in commercial packages of
therapeutic products, that contain information about the indications, usage, dosage,
administration, contraindications and/or warnings concerning the use of such
30 therapeutic products.
-30-
WO 01/74388 PCT/US01/10382
n. Production of Antagonists
The methods and articles of manufacture of the present invention use, or
incorporate, an antagonist which binds to a B cell surface marker and/or a cytokine.
5 Accordingly, methods for generating such antagonists will be described here. The B
cell surface marker or cytokine to be used for production of, or screening for,
antagonists) may be, e.g., a soluble form of the antigen or a portion thereof,
containing the desired epitope. Alternatively, or additionally, cells expressing the B
cell surface marker at their cell surface can be used to generate, or screen for,
10 antagonist(s). Other forms of the B cell surface marker useful for generating
antagonists will be apparent to those skilled in the art. Preferably, the B cell surface
marker is the CD 19 or CD20 antigen. Preferably, the cytokine is IL-10.
While the preferred antagonist is an antibody, antagonists other man
antibodies are contemplated herein. For example, the antagonist may comprise a
1 5 small molecule antagonist optionally fused to, or conjugated with, a cytotoxic agent
(such as those described herein). Libraries of small molecules may be screened against
the B cell surface marker of interest herein in order to identify a small molecule which
binds to that antigen. The small molecule may further be screened for its antagonistic
properties and/or conjugated with a cytotoxic agent.
20 The antagonist may also be a peptide generated by rational design or by phage
display (see, e.g., W098/35036 published 13 August 1998). In one embodiment, the
molecule of choice may be a "CDR mimic" or antibody analogue designed based on
the CDRs of an antibody. While such peptides may be antagonistic by themselves, the
peptide may optionally be fused to a cytotoxic agent so as to add or enhance
25 antagonistic properties of the peptide.
A description follows as to exemplary techniques for the production of the
antibody antagonists used in accordance with the present invention.
Polyclonal antibodies
Polyclonal antibodies are preferably raised in animals by multiple
30 subcutaneous (sc) or intraperitoneal (ip) injections of the relevant antigen and an
-31 -
WO 01/74388 PCT/USO 1/10382
adjuvant. It may be useful to conjugate the relevant antigen to a protein that is
immunogenic in the species to be immunized, e.g., keyhole limpet hemocyanin, serum
albumin, bovine thyroglobulin, or soybean trypsin inhibitor using a bifunctional or
derivatizing agent, for example, maleimidobenzoyl sulfosuccinimide ester
5 (conjugation through cysteine residues), N-hydroxysuccinimide (through lysine
residues), glutaraldehyde, succinic anhydride, SOC12, or R1N=C=NR, where R and
RI are different alkyl groups. Animals are immunized against the antigen,
immunogenic conjugates, or derivatives by combining, e.g., 100 pg or 5 wg of the
protein or conjugate (for rabbits or mice, respectively) with 3 volumes of Freund's
10 complete adjuvant and injecting the solution intradermally at multiple sites. One
month later the animals are boosted with 1 15 to 1/10 the original amount of peptide or
conjugate in Freund's complete adjuvant by subcutaneous injection at multiple sites.
Seven to 14 days later the animals are bled and the serum is assayed for antibody titer.
Animals are boosted until the titer plateaus. Preferably, the animal is boosted with the
15 conjugate of the same antigen, but conjugated to a different protein and/or through a
different cross-linking reagent. Conjugates also can be made in recombinant cell
culture as protein fusions. Also, aggregating agents such as alum are suitably used to
enhance the immune response.
(it) Monoclonal antibodies
20 Monoclonal antibodies are obtained from a population of substantially
homogeneous antibodies, Le., the individual antibodies comprising the population are
identical except for possible naturally occurring mutations that may be present in
minor amounts. Thus, the modifier "monoclonal" indicates the character of the
antibody as not being a mixture of discrete antibodies. For example, the monoclonal
25 antibodies may be made using the hybridoma method first described by Kohler et al,
Nature, 256:495 (1975), or may be made by recombinant DNA methods (U.S. Patent
No. 4,816,567).
In the hybridoma method, a mouse or other appropriate host animal, such as a
hamster, is immunized as hereinabove described to elicit lymphocytes that produce or
30 are capable of producing antibodies that will specifically bind to the protein used for
-32-
WO 01/74388 PCT/US01/10382
immunization. Alternatively, lymphocytes maybe immunized in vitro. Lymphocytes
then are fused with myeloma cells using a suitable fusing agent, such as polyethylene
glycol, to form a hybridoma
cell [Goding, Monoclonal Antibodies: Principles and Practice, pp.59-103 (Academic
5 Press, 1986)].
The hybridoma cells thus prepared are seeded and grown in a suitable culture
medium that preferably contains one or more substances that inhibit the growth or
survival of the unfused, parental myeloma cells. For example, if the parental myeloma
cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or
1 0 HPRT), the culture medium for the hybridomas typically will include hypoxanthine,
arninopterin, and thymidine (HAT medium), which substances prevent the growth of
HGPRT-deficient cells.
Preferred myeloma cells are those that fuse efficiently, support stable
high-level production of antibody by the selected antibody-producing cells, and are
15 sensitive to a medium such as HAT medium. Among these, preferred myeloma cell
lines are murine myeloma lines, such as those derived from MOPC-21 and MPC-1 1
mouse tumors available from the Salk Institute Cell Distribution Center, San Diego,
California USA, and SP-2 or X63-Ag8-653 cells available from the American Type
Culture Collection, Rockville, Maryland USA. Human myeloma and mouse human
20 heteromyeloma cell lines also have been described for the production of human
monoclonal antibodies [Kozbor, J. Immunol, 133:3001 (1984); Brodeur et al,
Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel
Dekker, Inc., New York, 1987)].
Culture medium in which hybridoma cells are growing is assayed for
25 production of monoclonal antibodies directed against the antigen. Preferably, the
binding specificity of monoclonal antibodies produced by hybridoma cells is
determined by immunoprecipitation or by an in vitro binding assay, such as
radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA). The
binding affinity of the monoclonal antibody can, for example, be determined by the
30 Scatchard analysis of Munson et al, Anal Biochem., 107:220 (1980).
-33-
WO 01/74388
PCT/US01/10382
After hybridoma cells are identified that produce antibodies of the desired
specificity, affinity, and/or activity, the clones maybe subcloned by limiting dilution
procedures and grown by standard methods (Goding, Monoclonal Antibodies:
Principles and Practice, pp.59-103 (Academic Press, 1986)). Suitable culture media
5 for this purpose include, for example, D-MEM or RPMI-1640 medium. In addition,
the hybridoma cells may be grown in vivo as ascites tumors in an animal.
The monoclonal antibodies secreted by the subclones are suitably separated
from the culture medium, ascites fluid, or serum by conventional immunoglobulin
purification procedures such as, for example, protein A-Sepharose, hydroxylapatite
10 chromatography, gel electrophoresis, dialysis, or affinity chromatography.
DNA encoding the monoclonal antibodies is readily isolated and sequenced
using conventional procedures (e.g., by using oligonucleotide probes that are capable
of binding specifically to genes encoding the heavy and light chains of murine
antibodies). The hybridoma cells serve as a preferred source of such DNA. Once
1 5 isolated, the DNA may be placed into expression vectors, which are then transfected
into host cells such as E. coli cells, simian COS cells, Chinese Hamster Ovary (CHO)
cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to
obtain the synthesis of monoclonal antibodies in the recombinant host cells. Review
articles on recombinant expression in bacteria of DNA encoding the antibody include
20 Skerra et ah, Curr. Opinion in Immunol., 5:256-262 (1993) and Phickthun, Immunol.
Revs., 130:151-188 (1992).
In a further embodiment, antibodies or antibody fragments can be isolated
from antibody phage libraries generated using the techniques described in McCafferty
et al, Nature, 348:552-554 (1990). Clackson et al, Nature, 352:624-628 (1991) and
25 Marks et al, J. Mol. Biol, 222:581-597 (1991) describe the isolation of murine and
human antibodies, respectively, using phage libraries. Subsequent publications
describe the production of high affinity (nM range) human antibodies by chain
shuffling (Marks et al, BiolTechnology, 10:779-783 (1992)), as well as combinatorial
infection and in vivo recombination as a strategy for constructing very large phage
30 libraries (Waterhouse et al, Nuc. Acids. Res., 21 :2265-2266 (1 993)). Thus, these
-34-
WO 01/74388 PCTYUS01/10382
techniques are viable alternatives to traditional monoclonal antibody hybridoma
techniques for isolation of monoclonal antibodies.
The DNA also may be modified, for example, by substituting the coding
sequence for human heavy- and light-chain constant domains in place of the
5 homologous murine sequences (U.S. Patent No. 4,8 1 6,567; Morrison, et al, Proc.
Natl Acad. Sci. USA, 81:6851 (1984)), or by covalently joining to the
immunoglobulin coding sequence all or part of the coding sequence for a
non-immunoglobuUn polypeptide. Typically such non-immunoglobulin polypeptides
are substituted for the constant domains of an antibody, or they are substituted for the
10 variable domains of one anu^en-combining site of an antibody to create a chimeric
bivalent antibody comprising one antigen-combining site having specificity for an
antigen and another antigen combining site having specificity for a different antigen.
(Hi) Humanized antibodies
Methods for humanizing non-human antibodies have been described in the art.
1 5 Preferably, a humanized antibody has one or more amino acid residues introduced into
it from a source which is non-human. These non-human amino acid residues are often
referred to as "import" residues, which are typically taken from an "import" variable
domain. Humanization can be essentially performed following the method of Winter
and co-workers (Jones et al, Nature, 321:522-525 (1986); Riechmann et al, Nature,
20 332:323-327 (1988); Verhoeyen et aL, Science, 239:1534-1536 (1988)), by
substituting hypervariable region sequences for the corresponding sequences of a
human antibody. Accordingly, such "humanized" antibodies are chimeric antibodies
(U.S. Patent No. 4,816,567) wherein substantially less than an intact human variable
domain has been substituted by the corresponding sequence from a non-human
25 species. In practice, humanized antibodies are typically human antibodies in which
some hypervariable region residues and possibly some FR residues are substituted by
residues from analogous sites in rodent antibodies.
The choice of human variable domains, both fight and heavy, to be used in
making the humanized antibodies is very important to reduce antigenicity. According
30 to the so-called "best-fit" method, the sequence of the variable domain of a rodent
-35-
WO 01/74388
PCT/US01/10382
antibody is screened against the entire library of known human variable-domain
sequences. The human sequence which is closest to that of the rodent is then accepted
as the human framework region (FR) for the humanized antibody (Sims et al, J.
Immunol, 151:2296 (1993); Chothia et al, J. Mol. Biol, 196:901 (1987)). Another
5 method uses a particular framework region derived from the consensus sequence of all
human antibodies of a particular subgroup of light or heavy chains. The same
framework may be used for several different humanized antibodies (Carter et aL,
Proc. Nad. Acad. Sci. USA, 89:4285 (1992); Presta et al, J. Immunol, 151:2623
(1993)).
10 It is further important that antibodies be humanized with retention of high
affinity for the antigen and other favorable biological properties. To achieve this goal,
according to a preferred method, humanized antibodies are prepared by a process of
analysis of the parental sequences and various conceptual humanized products using
three dimensional models of the parental and humanized sequences.
15 Three-dimensional immunoglobulin models are commonly available and are familiar
to those skilled in the art. Computer programs are available which illustrate and
display probable three-dimensional conformational structures of selected candidate
immunoglobulin sequences. Inspection of these displays permits analysis of the likely
role of the residues in the functioning of the candidate immunoglobulin sequence, i.e.,
20 the analysis of residues that influence the ability of the candidate immunoglobulin to
bind its antigen, hi Ibis way, FR residues can be selected and combined from the
recipient and import sequences so that the desired antibody characteristic, such as
increased affinity for the target antigen(s), is achieved. In general, the hypervariable
region residues are directly and most substantially involved in influencing antigen
25 binding.
(iv) Human antibodies
As an alternative to humanization, human antibodies can be generated. For
example, it is now possible to produce transgenic animals (e.g., mice) that are
capable, upon immunization, of producing a full repertoire of human antibodies in the
30 absence of endogenous immunoglobulin production. For example, it has been
-36-
WO 01/74388
PCT/US01/10382
described that the homozygous deletion of the antibody heavy-chain joining region
(JH) gene in chimeric and germ-line mutant mice results in complete inhibition of
endogenous antibody production. Transfer of the human germ-line immunoglobulin
gene array in such germ-line mutant mice will result in the production of human
5 antibodies upon antigen challenge. See, e.g., Jakobovits et al, Proc. Mad. Acad. Set
USA, 90:2551 (1993); Jakobovits et al, Nature, 362:255-258 (1993); Bruggermann et
al, Year inlmmuno., 7:33 (1993); and US Patent Nos. 5,591,669, 5,589,369 and
5,545,807. Alternatively, phage display technology (McCafferty etal, Nature
348:552-553 (1990)) can be used to produce human antibodies and antibody
10 fragments in vitro, from immunoglobulin variable (V) domain gene repertoires from
unimmunized donors. According to this technique, antibody V domain genes are
cloned in-frame into either a major or minor coat protein gene of a filamentous
bacteriophage, such as Ml 3 or fd, and displayed as functional antibody fragments oh
the surface of the phage particle. Because the filamentous particle contains a
1 5 single-stranded DNA copy of the phage genome, selections based on the functional
properties of the antibody also result in selection of the gene encoding the antibody
exhibiting those properties. Thus, the phage mimics some of the properties of the B
cell. Phage display can be performed in a variety of formats; for their review see, e.g.
Johnson, Kevin S. and Chiswell, David J., Current Opinion in Structural Biology
20 3:564-571(1993). Several sources of V-gene segments can be used for phage display.
Clackson et al, Nature, 352: 624-628 (1991) isolated a diverse array of
anti-oxazolone antibodies from a small random combinatorial library of V genes
derived from the spleens of immunized mice. A repertoire of V genes from
uinmmunized human donors can be constructed and antibodies to a diverse array of
25 antigens (including self-antigens) can be isolated essentially following the techniques
described by Marks et al, J. Mol Biol. 222:581-597 (1991), or Griffith et al, EMBO
J. 12:725-734 (1993). See, also, US Patent Nos. 5,565,332 and 5,573,905. Human
antibodies may also be generated by in vitro activated B cells (see US Patents
5,567,610 and 5,229,275).
30 (v) Antibody fragments
-37-
WO 01/74388 PCT/US01/10382
Various techniques have been developed for the production of antibody
fragments. Traditionally, these fragments were derived via proteolytic digestion of
intact antibodies (see, e.g., Morimoto et al., Journal of Biochemical and Biophysical
Methods 24:107-1 17 (1992) and Brennan et al, Science, 229:81 (1985)). However,
5 these fragments can now be produced directly by recombinant host cells. For example,
the antibody fragments can be isolated from the antibody phage libraries discussed
above. Alternatively, Fab'-Sli fragments can be directly recovered from E. coli and
chemically coupled to form F(ab')2 fragments [Carter et al, Bio/Technology
10:163-167 (1992)]. According to another approach, F(ab')2 fragments can be isolated
10 directly from recombinant host cell culture. Other techniques for the production of
antibody fragments will be apparent to the skilled practitioner. In other embodiments,
the antibody of choice is a single chain Fv fragment (scFv). See WO 93/16185; US
Patent No. 5,571,894; and US Patent No. 5,587,458. The antibody fragment may also
be a "linear antibody", e.g., as described in US Patent 5,641,870 for example. Such
1 5 linear antibody fragments may be monospecific or bispecific.
(vi) Bispecific antibodies
Bispecific antibodies are antibodies that have binding specificities for at least
two different epitopes. Exemplary bispecific antibodies may bind to two different
epitopes of the B cell surface marker. Other such antibodies may bind a first B cell
20 marker and further bind a second B cell surface marker. Alternatively, an anti-B cell
marker binding arm may be combined with an arm which binds to a triggering
molecule on a leukocyte such as a T-cell receptor molecule (e.g. CD2 or CD3), or Fc
receptors for IgG (FcyR), such as FcyPJ (CD64), FcyPJI (CD32) and FcyRm (CD 16)
so as to focus cellular defense mechanisms to the B cell. Bispecific antibodies may
25 also be used to localize cytotoxic agents to the B cell. These antibodies possess a B
cell marker-binding arm and an arm which binds the cytotoxic agent (e.g. saporin,
anti-interferon-a, vinca alkaloid, ricin A chain, methotrexate or radioactive isotope
hapten). Bispecific antibodies can be prepared as full length antibodies or antibody
fragments (e.g. F(ab')Z bispecific antibodies).
30 Methods for making bispecific antibodies are known in the art. Traditional
-38-
WO 01/74388
PCT/US01/10382
production of full length bispecific antibodies is based on the coexpression of two
immunoglobulin heavy chain-light chain pairs, where the two chains have different
specificities (Millstein et al., Nature, 305:537-539 (1983)). Because of the random
assortment of immunoglobulin heavy and light chains , these hybridomas (quadromas)
5 produce a potential mixture of 1 0 different antibody molecules, of which only one has
the correct bispecific structure. Purification of the correct molecule, which is usually
done by affinity chromatography steps, is rather cumbersome, and the product yields
are low. Similar procedures are disclosed in WO 93/08829, and in Traunecker et al,
EMBOJ, 10:3655-3659 (1991).
10 According to a different approach, antibody variable domains with the desired
binding specificities (antibody-antigen combining sites) are fused to immunoglobulin
constant domain sequences. The fusion preferably is with an immunoglobulin heavy
chain constant domain, comprising at least part of the hinge, CH2, and CH3 regions. It
is preferred to have the first heavy-chain constant region (CHI) containing the site
1 5 necessary for light chain binding, present in at least one of the fusions. DNAs
encoding the immunoglobulin heavy chain fusions and, if desired, the
immunoglobulin light chain, are inserted into separate expression vectors, and are
co-transfected into a suitable host organism. This provides for great flexibility in
adjusting the mutual proportions of the three polypeptide fragments in embodiments
20 when unequal ratios of the three polypeptide chains used in the construction provide
the optimum yields. It is, however, possible to insert the coding sequences for two or
all three polypeptide chains in one expression vector when the expression of at least
two polypeptide chains in equal ratios results in high yields or when the ratios are of
no particular significance.
25 m a preferred embodiment of this approach, the bispecific antibodies are
composed of a hybrid immunoglobulin heavy chain with a first binding specificity in
one arm, and a hybrid immunoglobulin heavy chain light chain pair (providing a
second binding specificity) in the other arm. It was found that this asymmetric
structure facilitates the separation of the desired bispecific compound from unwanted
30 immunoglobulin chain combinations, as the presence of an immunoglobulin light
-39-
WO 01/74388
PCT/US01/10382
chain in only one half of the bispecific molecule provides for a facile way of
separation. This approach is disclosed in WO 94/04690. For further details of
generating bispecific antibodies see, for example, Suresh et al, Methods in
Enzymology, 121:210(1986).
5 According to another approach described in US Patent No. 5,731,168, the
interface between a pair of antibody molecules can be engineered to maximize the
percentage of heterodimers which are recovered from recombinant cell culture. The
preferred interface comprises at least a part of the CH3 domain of an antibody
constant domain. In this method, one or more small amino acid side chains from the
1 0 interface of the first antibody molecule are replaced with larger side chains (e.g.
tyrosine or tryptophan). Compensatory "cavities" of identical or similar size to the
large side chain(s) are created on the interface of the second antibody molecule by
replacing large amino acid side chains with smaller ones (e.g. alanine or threonine).
This provides' a mechanism for increasing the yield of the heterodimer over other
15 unwanted end-products such as homodimers.
Bispecific antibodies include cross-linked or "heteroconjugate" antibodies. For
example, one of the antibodies in the heteroconjugate can be coupled to avidin, the
other to biotin. Such antibodies have, for example, been proposed to target immune
system cells to unwanted cells (US Patent No. 4,676,980), and for treatment of HIV
20 infection (WO 91/00360, WO 92/200373, and EP 03089). Heteroconjugate antibodies
may be made using any convenient cross-linking methods. Suitable cross-linking
agents are well known in the art, and are disclosed in US Patent No. 4,676,980, along
with a number of cross-linking techniques.
Techniques for .generating bispecific antibodies from antibody fragments have
25 also been described in the literature. For example, bispecific antibodies can be
prepared using chemical linkage. Brennan et al, Science, 229:81 (1985) describe a
procedure wherein intact antibodies are proteolytically cleaved to generate F(ab*)2
fragments. These fragments are reduced in the presence of the dithiol complexing
agent sodium arsenite to stabilize vicinal dithiols and prevent intermolecular disulfide
30 formation. The Fab' fragments generated are then converted to thionitrobenzoate
-40-
WO 01/74388
PCT/US01/10382
(TNB) derivatives. One of the Fab'-TNB derivatives is then reconverted to the
Fab'-thiol by reduction with mercaptoethylamine and is mixed with an equirnolar
amount of the other Fab'-TNB derivative, to form the bispecific antibody. The
bispecific antibodies produced can be used as agents for the selective immobilization
5 of enzymes.
Recent progress has facilitated the direct recovery of Fab'-SH fragments from
E. coli, which can be chemically coupled to form bispecific antibodies. Shalaby et al,
J. Exp. Med., 175: 217-225 (1992) describe the production of a fully humanized
bispecific antibody F(ab')2 molecule. Each Fab' fragment was separately secreted from
10 E. coli and subjected to directed chemical coupling in vitro to form the bispecific
antibody. The bispecific antibody thus formed was able to bind to cells
overexpressing the ErbB2 receptor and normal human T cells, as well as trigger the
lytic activity of human cytotoxic lymphocytes against human breast tumor targets.
Various techniques for making and isolating bispecific antibody fragments
1 5 directly from recombinant cell culture have also been described. For example,
bispecific antibodies have been produced using leucine zippers. Kostelny et al, J.
Immunol, 148(5): 1547-1 553 (1992). The leucine zipper peptides from the Fos and
Jun proteins were linked to the Fab' portions of two different antibodies by gene
fusion. The antibody homodimers were reduced at the hinge region to form monomers
20 and then re-oxidized to form the antibody heterodimers. This method can also be
utilized for the production of antibody homodimers. The "diabody" technology
described by Hollinger et al, Proc. Natl Acad. Sci. USA, 90:6444-6448 (1993) has
provided an alternative mechanism for making bispecific antibody fragments. The
fragments comprise a heavy-chain variable domain (VH) connected to a light-chain
25 variable domain (VL) by a linker which is too short to allow pairing between the two
domains on the same chain.
Accordingly, the VH arid VL domains of one fragment are forced to pair with
the complementary VL and VH domains of another fragment, thereby forming two
antigen-binding sites. Another strategy for making bispecific antibody fragments by
30 the use of single-chain Fv (sFv) dimers has also been reported. See Gruber et al, J.
-41 -
WO 01/74388 PCTYUS01/10382
Immunol, 152:5368 (1994). Antibodies with more than two valencies are
contemplated. For example, trispecific antibodies can be prepared. Tutt et al. J.
Immunol. 147: 60 (1991).
m. Conjugates and Other Modifications of the Antagonist
5 The antagonists used in the methods or included in the articles of manufacture
herein are optionally conjugated to a cytotoxic agent. Chemotherapeutic agents useful
in the generation of such antagonist-cytotoxic agent conjugates have been described
above.
Conjugates of an antagonist and one or more small molecule toxins, such as a
10 cahcheamicin, a maytansine (US Patent No. 5,208,020), a trichothene, and CC1065
are also contemplated herein. In one embodiment of the invention, the antagonist is
conjugated to one or more maytansine molecules (e.g. about 1 to about 10
maytansinemolecules per antagonist molecule). Maytansine may, for example, be
converted to May-SS-Me which may be reduced to May-SH3 and reacted with
15 modified antagonist (Chari et al. Cancer Research 52: 127-131 (1992)) to generate a
maytansinoid-antagonist conjugate.
Alternatively, the antagonist is conjugated to one or more calicheamicin
molecules. The calicheamicin family of antibiotics are capable of producing
double-stranded DNA breaks at sub-picomolar concentrations. Structural analogues
20 of calicheamicin which may be used include, but are not limited to, 'yJ 1 , a2 1 , a3 1 ,
N-acetyl-yl", PSAG and 01 1 (Hinman et al. Cancer Research 53: 3336-3342 (1993)
and Lode et al. Cancer Research 58: 2925-2928 (1998)).
Enzymatically active toxins and fragments thereofwhich can be used include
diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain
25 (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain,
alpha-sarcin, 41euritesfordii proteins, dianthin proteins, Phytolaca americana proteins
(PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria
officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin and the
tricothecenes. See, for example, WO 93/21232 published October 28, 1993.
30 The present invention further contemplates antagonist conjugated with a
-42-
WO 01/74388 PCTAJS01/10382
compound with nucleolytic activity (e.g. a ribonuclease or a DNA endonuclease such
as a deoxyribonuclease; DNase). A variety of radioactive isotopes are available for
the production of radioconjugated antagonists. Examples include Ate",113',1125, Y9o
Re 186, Re 188, Sml53, Bi212 P32 and radioactive isotopes of Lu. Conjugates of the
5 antagonist and cytotoxic agent may be made using a variety of bifunctional protein
coupling agents such as N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP),
succinimidyl-4-(N-maleimidomethyl) cyclohexane-l-carboxylate, iminothiolane (IT),
bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCL), active
esters (such as disuccinimidyl suberate), aidehydes (such as glutareldehyde), bis azido
10 compounds (such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives
(such as bis-(pdiazoniumbenzoyl)-emylenediamine), diisocyanates (such as tolyene
2,6-diisocyanate), and bis-active fluorine compounds (such as
l,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin can be prepared
as described in Vitetta et al. Science 238:1098 (1987). Carbon- 14-labeled
15 l-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid (MX-DTPA) is
an exemplary chelating agent for conjugation of radionucleotide to the antagonist. See
W094/1 1026. The linker may be a "cleavable linker" facilitating release of the
cytotoxic drug in the cell. For example, an acid-labile linker, peptidase-sensitive
linker, dimethyl linker or msmfide-containing linker (Chari et ah Cancer Research
20 52: 127-13 1 (1992)) may be used. Alternatively, a fusion protein comprising the
antagonist and cytotoxic agent may be made, e.g. by recombinant techniques or
peptide synthesis.
In yet another embodiment, the antagonist may be conjugated to a "receptor"
(such streptavidin) for utilization in tumor pretargeting wherein the
25 antagonist-receptor conjugate is administered to the patient, followed by removal of
unbound conjugate from the circulation using a clearing agent and then administration
of a "ligand" (e.g. avidin) which is conjugated to a cytotoxic agent (e.g. a
radionucleotide). The antagonists of the present invention may also be conjugated
with a prodrug-activating enzyme which converts a prodrug (e.g. a pepndyl
30 chemotherapeutic agent, see W081/01 145) to an active anti-cancer drug. See, for
-43-
WO 01/74388
PCTYUS01/10382
example, WO 88/07378 and U.S. Patent No. 4,975,278.
The enzyme component of such conjugates includes any enzyme capable of
acting on a prodrug in such a way so as to covert it into its more active, cytotoxic
form. Enzymes that are useful in the method of this invention include, but are not
5 limited to, alkaline phosphatase useful for converting phosphate-cOntaining prodrugs
into free drugs; arylsulfatase useful for converting sulfate containing prodrugs into
free drugs; cytosine deaminase useful for converting non-toxic 5-fluorocytosine into
the anti-cancer drug, 5-fluorouracil; proteases, such as serratia protease, thermolysin,
subtilisin, carboxypeptidases and cathepsins (such as cathepsins B and L), that are
10 useful for converting peptide-containing prodrugs into free drugs;
D-alanylcarboxypeptidases, useful for converting prodrugs that contain D-amino acid
substituents; carbohydrate cleaving enzymes such as li-galactosidase and
neuraminidase useful for converting glycosylated prodrugs into free drugs;
(3-lactamase useful for converting drugs derivatized with (3-lactams into free drugs;
1 5 and penicillin amidases, such as penicillin V amidase or penicillin G amidase, useful
for converting drugs derivatized at their amine nitrogens with phenoxyacetyl or
phenylacetyl groups, respectively, into free drugs. Alternatively, antibodies with
enzymatic activity, also known in the art as "abzymes", can be used to convert the
prodrugs of the invention into free active drugs (see, e.g., Massey, Nature 328:
20 457-458 (1987)). Antagonist-abzyme conjugates can be prepared as described herein
for delivery of the abzyme to a tumor cell population.
The enzymes of this invention can be covalently bound to the antagonist by
techniques well known in the art such as the use of the heterobifunctional crosslinking
reagents discussed above. Alternatively, fusion proteins comprising at least the
25 antigen binding region of an antagonist of the invention linked to at least a
functionally active portion of an enzyme of the invention can be constructed using
recombinant DNA techniques well known in the art [see, e.g., Neuberger et al,
Nature, 312: 604-608 (1984)].
Other modifications of the antagonist are contemplated herein. For example,
30 the antagonist may be linked to one of a variety of nonproteinaceous polymers, e.g.,
-44-
WO 01/74388
PCT/US01/10382
polyethylene glycol, polypropylene glycol, polyoxyalkylenes, or copolymers of
polyethylene glycol and polypropylene glycol. The antagonists disclosed herein may
also be formulated as liposomes. Liposomes containing the antagonist are prepared by
methods known in the art, such as described in Epstein et al, Proc. Mad. Acad Sci.
5 USA, 82:3688 (1985); Hwang et al, Proc. Natl Acad. Sci. USA, 77:4030 (1980); U.S.
Pat. Nos. 4,485,045 and 4,544,545; and W097/38731 published October 23, 1997.
Liposomes with enhanced circulation time are disclosed in U.S. Patent No. 5,013,556.
Particularly useful liposomes can be generated by the reverse phase
evaporation method with a lipid composition comprising phosphatidylcholine,
0 cholesterol and PEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes are
extruded through filters of defined pore size to yield liposomes with the desired
diameter. Fab' fragments of an antibody of the present invention can be conjugated to
the liposomes as described in Martin et al, J. Biol. Chem. 257: 286-288 (1982) via a
disulfide interchange reaction. A chemotherapeutic agent is optionally contained
5 within the liposome. See Gabizon et al. J. National Cancer Inst.81(19)1484 (1989).
Amino acid sequence modifications) of protein or peptide antagonists described
herein are contemplated. For example, it may be desirable to improve the binding
affinity and/or other biological properties of the antagonist.
Amino acid sequence variants of the antagonist are prepared by introducing
0 appropriate nucleotide changes into the antagonist nucleic acid, or by peptide
synthesis. Such modifications include, for example, deletions from, and/or insertions
into and/or substitutions of, residues within the amino acid sequences of the
antagonist. Any combination of deletion, insertion, and substitution is made to arrive
at the final construct, provided that the final construct possesses the desired
5 characteristics. The amino acid changes also may alter post-translational processes of
the antagonist, such as changing the number or position of glycosylation sites.
A useful method for identification of certain residues or regions of the
antagonist that are preferred locations for mutagenesis is called "alanine scanning
mutagenesis" as described by Cunningham and Wells Science, 244:1081-1085 (1989).
0 Here, a residue or group of target residues are identified (e.g., charged residues such
-45-
WO 01/74388
PCT/US01/10382
as arg, asp, his, lys, and glu) and replaced by a neutral or negatively charged amino
acid (most preferably alanine or polyalanine) to affect the interaction of the amino
acids with antigen. Those amino acid locations demonstrating functional
sensitivity to the substitutions then are refined by introducing further or other variants
5 at, or for, the sites of substitution. Thus, while the site for introducing an amino acid
sequence variation is predetermined, the nature of the mutation per se need not be
predeterrnined. For example, to analyze the performance of a mutation at a given site,
ala scanning or random mutagenesis is conducted at the target codon or region and the
expressed antagonist variants are screened for the desired activity.
10 Amino acid sequence insertions include amino- and/or carboxyl-terminal
fusions ranging in length from one residue to polypeptides containing a hundred or
more residues, as well as intrasequence insertions of single or multiple amino acid
residues. Examples of terminal insertions include an antagonist with an N-terminal
methionyl residue or the antagonist fused to a cytotoxic polypeptide. Other insertional
1 5 variants of the antagonist molecule include the fusion to the N- or C-terminus of the
antagonist of an enzyme, or a polypeptide which increases the serum half-life of the
antagonist.
Another type of variant is an amino acid substitution variant. These variants
have at least one amino acid residue in the antagonist molecule replaced by different
20 residue. The sites of greatest interest for substitutional mutagenesis of antibody
antagonists include the hypervariable regions, but FR alterations are also
contemplated.
Conservative substitutions are shown in Table 1 under the heading of
"preferred substitutions". If such substitutions result in a change in biological activity,
25 then more substantial changes, denominated "exemplary substitutions" in Table 1 , or
as further described below in reference to arnino acid classes, may be introduced and
the products screened.
Table 1
Original
Exemplary
Preferred
Residue
Substitutions
Substitutions
-46-
WO 01/74388
PCT/US01/10382
Ala (A)
val; leu; ile
val
Arg(R)
lys; gin; asn
lys
Asn(N)
gin; his; asp, lys; arg
gin
Asp(D)
glu; asn
glu
Cys(C)
ser; ala
ser
Gin CO)
asn; glu
asn
Glu (E)
asp; gin
asp
Gly(G)
ala
ala
His (H)
asn; gin; lys; arg
arg
He (I)
leu; val; met; ala;
phe; norleucine
ICU
Lea (L)
norleucine; ile; val;
met; ala; phe
ile
Lys(K)
arg; gin; asn
arg
Met(M)
leu; phe; ile
leu
Phe(F)
leu; val; ile; ala; tyr
tyr
Pro(P)
ala
ala
Ser (S)
thr
thr
Thr(T)
ser
ser
TIP (W)
tyr; phe
tyr
Tyr(Y)
trp; phe; thr; ser
phe
Val(V)
ile; leu; met; phe;
ala; norleucine
ICU
Substantial modifications in the biological properties of the antagonist are
accomplished by selecting substitutions that differ significantly in their effect on
mamtaining (a) the structure of the polypeptide backbone in the area of the
5 substitution, for example, as a sheet or helical conformation, (b) the charge or
hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain.
Naturally occurring residues are divided into groups based on common side-chain
properties:
(1) hydrophobic: norleucine, met, ala, val, leu, ile;
10 (2) neutral hydrophilic: cys, ser, thr;
(3) acidic: asp, glu;
(4) basic: asn, gin, his, lys, arg;
(5) residues that influence chain orientation: gly, pro; and
(6) aromatic: trp, tyr, phe.
-47-
WO 01/74388
PCT/US01/10382
Non-conservative substitutions will entail exchanging a member of one of
these classes for another class.
Any cysteine residue not involved in maintaining the proper conformation of
the antagonist also may be substituted, generally with serine, to improve the oxidative
5 stability of the molecule and prevent aberrant crosslinking. Conversely, cysteine
bond(s) may be added to the antagonist to improve its stability (particularly where the
antagonist is an antibody fragment such as an Fv fragment).
A particularly preferred type of substitutional variant involves substituting one
or more hypervariable region residues of a parent antibody. Generally, the resulting
10 variant(s) selected for further development will have improved biological properties
relative to the parent antibody from which they are generated. A convenient way for
generating such substitutional variants is affinity maturation using phage display.
Briefly, several hypervariable region sites (e.g.6-7 sites) are mutated to generate all
possible amino substitutions at each site. The antibody variants thus generated are
1 5 displayed in a monovalent fashion from filamentous phage particles as fusions to the
gene m product of Ml 3 packaged within each particle. The phage-displayed variants
are then screened for their biological activity (e.g. binding affinity) as herein
disclosed. In order to identify candidate hypervariable region sites for modification,
alanine scanning mutagenesis can be performed to identify hypervariable region
20 residues contributing significantly to antigen binding. Alternatively, or in additionally,
it may be beneficial to analyze a crystal structure of the antigen-antibody complex to
identify contact points between the antibody and antigen. Such contact residues and
neighboring residues are candidates for substitution according to the techniques
elaborated herein. Once such variants are generated, the panel of variants is subjected
25 to screening as described herein and antibodies with superior properties in one or
more relevant assays may be selected for further development.
Another type of amino acid variant of the antagonist alters the original
glycosylation pattern of the antagonist. By altering is meant deleting one or more
carbohydrate moieties found in the antagonist, and/or adding one or more
30 glycosylation sites that are not present in the antagonist.
-48-
WO 01/74388 PCT/US01/10382
Glycosylation of polypeptides is typically either N-linked or O-linked.
N-linked refers to the attachment of the carbohydrate moiety to the side chain of an
asparagine residue. The tripeptide sequences asparagine-X-serine and
asparagine-X-threonine, where X is any amino acid except proline, are the recognition
5 sequences for enzymatic attachment of the carbohydrate moiety to the asparagine side
chain. Thus, the presence of either of these tripeptide sequences in a polypeptide
creates a potential glycosylation site. O-linked glycosylation refers to the attachment
of one of the sugars N-aceylgalactosamine, galactose, or xylose to a hydroxyamino
acid, most commonly serine or threonine, although 5-hydroxyproline or
10 5-hydroxylysine may also be used. Addition of glycosylation sites to the antagonist is
conveniently accomplished by altering the amino acid sequence such that it contains
one or more of the above-described tripeptide sequences (for N-linked glycosylation
sites). The alteration may also be made by the addition of, or substitution by, one or
more serine or threonine residues to the sequence of the original antagonist (for
15 O-linked glycosylation sites).
Nucleic acid molecules encoding amino acid sequence variants of the
antagonist are prepared by a variety of methods known in the art. These methods
include, but are not limited to, isolation from a natural source (in the case of naturally
occurring amino acid sequence variants) or preparation by oligonucleotide-mediated
20 (or site directed) mutagenesis, PCR mutagenesis, and cassette mutagenesis of an
earlier prepared variant or a non-variant version of the antagonist.
It may be desirable to modify the antagonist of the invention with tespect to
effector function, e.g. so as to enhance antigen-dependent cell-mediated cyotoxicity
(ADCC) and/or complement dependent cytotoxicity (CDC) of the antagonist. This
25 may be achieved by introducing one or more amino acid substitutions in an Fc region
of an antibody antagonist. Alternatively or additionally, cysteine residue(s) may be
introduced in the Fc region, thereby allowing interchain disulfide bond formation in
this region. The homodimeric antibody thus generated may have improved
internalization capability and/or increased complement-mediated cell killing and
30 antibody-dependent cellular cytotoxicity (ADCC). See Caron et al, J. Exp Med.
-49-
WO 01/74388 PCT/US01/10382
176:1191-1195 (1992) and Shopes, B. J. Immunol. 148:2918-2922 (1992).
Homodimeric antibodies with enhanced anti-tumor activity may also be prepared
using heterobifunetional cross-linkers as described in Wolff et al. Cancer Research
53:2560-2565 (1993). Alternatively, an antibody can be engineered which has dual Fc
5 regions and may thereby have enhanced complement lysis and
ADCC capabilities. See Stevenson et al Anti-Cancer Drug Design 3:219-230 (1989).
To increase the serum half life of the antagonist, one may incorporate a
salvage receptor binding epitope into the antagonist (especially an antibody fragment)
as described in US Patent 5,739,277, for example. As used herein, the term "salvage
10 receptor binding epitope" refers to an epitope of the Fc region of an IgG molecule
(e.g., IgGl, IgG2, IgG3, or IgG4) that is responsible for increasing the in vivo serum
half-life of the IgG molecule.
IV. Pharmaceutical Formulations
Therapeutic formulations of the antagonists used in accordance with the
1 5 present invention are prepared for storage by mixing an antagonist or antagonists
having the desired degree of purity with optional pharmaceutically acceptable carriers,
excipients or stabilizers (Remington's Pharmaceutical Sciences 16th edition, Osol, A.
Ed. (1980)), in the form of lyophilized formulations or aqueous solutions. Acceptable
carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and
20 concentrations employed, and include buffers such as phosphate, citrate, and other
organic acids; antioxidants including ascorbic acid and methionine; preservatives
(such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;
benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl
parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol;
25 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues)
polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins;
hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine,
glulamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides,
and other carbohydrates including glucose, mannose, or dextrins; chelating agents
30 such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt forming
-50-
WO 01/74388 PCT/US01/10382
counter-ions such as sodium; metal complexes (e.g. Zn-protein complexes); and/or
non-ionic surfactants such as TWEENTM, PLURONICSTM or polyethylene glycol
(PEG).
Exemplary anti-CD20 antibody formulations are described in W098/56418,
5 expressly incorporated herein by reference. This publication describes a liquid
multidose formulation comprising 40 mg/mL rituximab, 25 mM acetate, 150 mM
trehalose, 0.9% benzyl alcohol, 0.02% polysorbate 20 at pH 5.0 that has a minimum
shelf life of two years storage at 2-8 °C. Another anti-CD20 formulation of interest
comprises 1 Omg/mL rituximab in 9.0 mg/mL sodium chloride, 7.35 mg/mL sodium
10 citrate dihydrate, 0.7mg/mL polysorbate 80, and Sterile Water for Injection, pH 6.5.
Lyophilized formulations adapted for subcutaneous administration are described in
W097/04801. Such lyophilized formulations may be reconstituted with a suitable
diluent to a high protein concentration and the reconstituted formulation may be
administered subcutaneously to the mammal to be treated herein.
1 5 The formulation herein may also contain more than one active compound zi.;
necessary for the particular indication being treated, preferably those with
complementary activities that do not adversely affect each other. For example, it may
be desirable to further provide a cytotoxic agent, chemotherapeutic agent, cytokine or
immunosuppressive agent (e.g. one which acts on T cells, such as cyclosporin or an
20 antibody that binds T cells, e.g. one which binds LFA-1). The effective amount of
such other agents depends on the amount of antagonist present in the formulation, the
type of disease or disorder or treatment, and other factors discussed above. These are
generally used in the same dosages and with a<iministration routes as used
hereinbefore or about from 1 to 99% of the heretofore employed dosages.
25 The active ingredients may also be entrapped in microcapsules prepared, for
example, by coacervation techniques or by interfacial polymerization, for example,
hydroxymethylcellulose or gelatin-microcapsules and poly(methylmethacylate)
microcapsules, respectively, in colloidal drug delivery systems (for example,
liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules)
30 or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical
-51-
WO 01/74388
PCT/US01/10382
Sciences 1 6th edition, Osol, A. Ed. (1 980).
Sustained-release preparations maybe prepared. Suitable examples of
sustained-release preparations include semipermeable matrices of solid hydrophobic
polymers containing the antagonist, which matrices are in the form of shaped articles,
5 e.g. films, or microcapsules. Examples of sustained-release matrices include
polyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate), or
poly(vinylalcohol)), polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic
acid and y ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic
acid-glycolic acid copolymers such as the LUPRON DEPOTTM (injectable
10 microspheres composed of lactic acid glycolic acid copolymer and leuprolide acetate),
and poly-D-(-)-3-hydroxybutyric acid.
The formulations to be used for in vivo administration must be sterile. This is
readily accomplished by filtration through sterile filtration membranes.
15 V. Treatment with the Antagonist
A composition comprising an antagonist which binds to a B cell surface
antigen and a composition which contains a cytokine antagonist, e.g. an antibody,
wherein both may be in the same composition will be formulated, dosed, and
administered in a fashion consistent with good medical practice. Preferably, the anti-
20 cytokine will comprise an anti-ILlO antibody and the B cell antagonist will comprise a
B cell depleting antibody, preferably an anti-CD20 antibody such as Rituxan®.
Factors for consideration in this context include the particular disease or disorder
being treated, the particular mammal being treated, the clinical condition of the
individual patient, the cause of the disease or disorder, the site of delivery of the
25 agent, the method of administration, the scheduling of administration, and other
factors known to medical practitioners. The therapeutically effective amount of the
antagonist to be administered will be governed by such considerations.
As a general proposition, the therapeutically effective amount of the antagonist
administered parenterally per dose will be in the range of about 0.1 to 20 mg/kg of
30 patient body weight per day, with the typical initial range of antagonist used being in
-52-
WO 01/74388
PCT/US01/10382
the range of about 2 to 10 mg/kg.
The preferred antagonist is an antibody, e.g. an antibody such as RITUXAN®,
which is not conjugated to a cytotoxic agent. Suitable dosages for an unconjugated
antibody are, for example, in the range from about 20mg/mz to about I OOOmg/m2.
5 In one embodiment, the dosage of the antibody differs from that presently
recommended for RITUXAN®. For example, one may administer to the patient one
or more doses of substantially less than 375mg/m2 of the antibody, e.g. where the
dose is in the range from about 20mg/mz to about 250mg/m2, for example from about
50mg/m2 to about 200mg/m2.
10 Moreover, one may administer one or more initial dose(s) of the antibody
followed by one or more subsequent dose(s), wherein the mg/m2 dose of the antibody
in the subsequent dose(s) exceeds the mg/m2 dose of the antibody in the initial
dose(s). For example, the initial dose may be in the range from about 20mg/m2 to
about 250mg/m2 (e.g. from about 50mg/m2 to about 200mg/mz) and the subsequent
15 dose may be in the range from about 250mg/m2 to about 1000mg/m2.
As noted above, however, these suggested amounts of antagonist are subject to
a great deal of therapeutic discretion. The key factor in selecting an appropriate dose
and scheduling is the result obtained, as indicated above.
For example, relatively higher doses may be needed initially for the treatment
20 of ongoing and acute diseases. To obtain the most efficacious results, depending on
the disease or disorder, the antagonist is administered as close to the first sign,
diagnosis, appearance, or occurrence of the disease or disorder as possible or during
remissions of the disease or disorder.
The antagonist is administered by any suitable means, including parenteral,
25 subcutaneous, intraperitoneal, intrapulmonary, and intranasal, and, if desired for local
immunosuppressive treatment, intralesional administration. Parenteral infusions
include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous
administration.
In addition, the antagonist may suitably be administered by pulse infusion,
30 e.g., with declining doses of the antagonist. Preferably the dosing is given by
-53-
WO 01/74388
PCT/US01/10382
injections, most preferably intravenous or subcutaneous injections, depending in part
on whether the administration is brief or chronic.
One may administer other compounds, such as cytotoxic agents,
chemotherapeutic agents, immunosuppressive agents and/or cytokines with the
5 antagonists herein. The combined administration includes coadministration, using
separate formulations or a single pharmaceutical formulation, and consecutive
administration in either order, wherein preferably there is a time period while both (or
all) active agents simultaneously exert their biological activities.
Aside from administration of protein antagonists to the patient the present ■
1 0 application contemplates administration of antagonists by gene therapy. Such
administration of nucleic acid encoding the antagonist is encompassed by the
expression "administering a therapeutically effective amount of an antagonist". See,
for example, W096/07321 published March 14, 1996 concerning the use of gene
therapy to generate intracellular antibodies.
1 5 There are two major approaches to getting the nucleic acid (optionally
contained in a vector) into the patient's cells; in vivo and ex vivo. For in vivo delivery
the nucleic acid is injected directly into the patient, usually at the site where the
antagonist is required. For ex vivo treatment, the patient's cells are removed, the
nucleic acid is introduced into these isolated cells and the modified cells are
20 adrninistered to the patient either directly or, for example, encapsulated within porous
membranes which are implanted into the patient (see, e.g. U.S. Patent Nos. 4,892,538
and 5,283,187). There are a variety of techniques available for introducing nucleic
acids into viable cells. The techniques vary depending upon whether the nucleic acid
is transferred into cultured cells in vitro, or in vivo in the cells of the intended host.
25 Techniques suitable for the transfer of nucleic acid into mammalian cells in vitro
include the use of liposomes, electroporation, microinjection, cell fusion,
DEAE-dextran, the calcium phosphate precipitation method, etc. A commonly used
vector for ex vivo delivery of the gene is a retrovirus.
The currently preferred in vivo nucleic acid transfer techniques include
30 transfection with viral vectors (such as adenovirus, Herpes simplex I virus, or
-54-
WO 01/74388 PCT/US01/10382
J
adeno-associated virus) and lipid-based systems (useful lipids for lipid mediated
transfer of the gene are DOTMA, DOPE and DC-Choi, for example). In some
situations it is desirable to provide the nucleic acid source with an agent that targets
the target cells, such as an antibody specific for a cell surface membrane protein or the
5 target cell, a ligand for a receptor on the target cell, etc. Where liposomes are
employed, proteins which bind to a cell surface membrane protein associated with
endocytosis may be used for targeting and/or to facilitate uptake, e.g. capsid proteins
or fragments thereof tropic for a particular cell type, antibodies for proteins which
undergo internalization in cycling, and proteins that target intracellular localization
10 and enhance intracellular half-life. The technique of receptor-mediated endocytosis is
described, for example, by Wu et aL, J. Biol. Chem. 262:4429-4432 (1987); and
Wagner et aL, Proc. Nad. Acad. Sci. USA 87:3410-3414 (1990). For review of the
currently known gene marking and gene therapy protocols see Anderson et aL,
Science 256:808-813 (1992). See also WO 93/25673 and the references cited therein.
15 VI. Articles of Manufacture
In another embodiment of the invention, an article of manufacture containing
materials useful for the treatment of the diseases or disorders described above is
provided. The article of manufacture comprises a container and a label or package
insert on or associated with the container. Suitable containers include, for example,
20 bottles, vials, syringes, etc. The containers maybe formed from a variety of materials
such as glass or plastic. The container holds or contains a composition which is
effective for treating the disease or disorder of choice and may have a sterile access
port (for example the container may be an intravenous solution bag or a vial having a
stopper pierceable by a hypodermic injection needle). At least one active agent in the
25 composition is the antagonist which binds a B cell surface marker. Preferably CD20,
and an anti-cytokine antibody, e.g. an anti-ILlO antibody. The label or package insert
indicates that the composition is used for treating a patient having or predisposed to
an autoimmune disease, such as those listed herein. The article of manufacture may
further comprise a second container comprising a pharmaceutically- acceptable
30 diluent buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered
-55 -
WO 01/74388
PCT/US01/10382
saline, Ringer's solution and dextrose solution. It may further include other materials
desirable from a commercial and user standpoint, including other buffers, diluents,
filters, needles, and syringes.
Further details of the invention are illustrated by the following non-limiting
5 Examples. The disclosure of all citations in the specification are expressly
incorporated by reference.
. Examples
10 Example 1
Treatment of Non-Hodgkin's Lymphoma
A patient with non-Hodgkin's lymphoma is intravenously administered an
anti-DLlO antibody at a dosage of 50mg/m 2 IV weekly for four weeks. Thereafter, the
patient is administered RITUXAN® intravenously according to the following dosage
15 schedules:
(A) 50mg/m 2 IV day 1
1 50mg/m 2 IV days on 8, 1 5 & 22
(B) 150mg/m 2 rVdayl
20 375mg/m 2 IV on days 8, 15 & 22
(C) 375mg/m 2 IV on days 1, 8, 15 & 22
This same patient is administered CHOP chemotherapy according to the
regimen described in US Patent 5,736,137.
25 After treatment, the patient is monitored to evaluate the effect on lymphoma
status, e.g., number and size of tumors.
Example 2
Treatment of Solid Tumor in Advanced Stage
30 A patient having an advanced colorectal cancer characterized by B cell
involvement is treated concurrently with an anti-ILlO antibody and RITUXAN® at
the same dosages as in Example 1 .
-56-
WO 01/74388
PCT/US01/10382
After treatment the patient is evaluated to determine whether such treatment
has resulted in an anti-tumor response, e.g., based on tumor shrinkage, lower tumor
antigen expression or other means of evaluating disease prognosis.
-57-
WO 01/74388
PCT/US01/10382
WHAT IS CLAIMED :
1 . A method of avoiding, decreasing or overcoming the resistance of
hematologic malignant cells or solid non-hematologic tumor cells to at least one
chemotherapeutic agent, comprising administering an anti-cytokine antibody or
5 fragment thereof or cytokine antagonist to a patient diagnosed with a hematologic
malignancy or a solid, non-hematologic tumor prior, concurrent or after
administration of at least one chemotherapeutic agent.
2. The method of Claim 1, wherein said hematologic cells comprise B
10 cell lymphoma or leukemia cells.
3 . The method of Claim 2, wherein said B cell lymphoma is selected from
the group consisting of low grade/ follicular non-Hodgkin' s lymphoma (NHL), small
lymphocytic (SL) NHL, intermediate grade/ follicular NHL, intermediate grade
15 diffuse NHL, high grade immunoblastic NHL, high grade lymphoblastic NHL, high
grade small non-cleaved cell NHL, bulky disease NHL and Waldenstrom's
Macroglobulinemia.
4. The method of Claim 3, wherein said B cell lymphoma is low grade/
20 follicular non-Hodgkins lymphoma (NHL).
5. The method of Claim 2, wherein said leukemic cell is selected from the
group consisting of acute lymphoblastic leukemia, acute myelogenous leukemia,
chronic lymphocytic leukemia, chronic myelogenous leukemia, lymphoblastic
25 leukemia, lymphocytic leukemia, monocytic leukemia, myelogenous leukemia, and
promyelocyte leukemia.
6. The method of Claim 1, wherein said at least one chemotherapeutic
agent is selected from the group consisting of CHOP, ICE, Mitozantrone, Cytarabine,
30 DVP, ATRA, Idarubicin, hoelzer chemotherapy regime, La La chemotherapy regime,
-58-
WO 01/74388
PCT/US01/10382
ABVD, CEOP, 2-CdA, FLAG & IDA with or without subsequent G-CSF treatment),
VAD, M & P, C-Weekly, ABCM, MOPP, DHAP, daunorubicin, doxorubicin,
tamoxifen, toremifene, methotrexate, and cisplatin.
5 7. The method of Claim 1, wherein said cytokine is selected from the
group consisting of JL2, IL6, IL10 and TNF-alpha.
8. The method of Claim 7, wherein said cytokine is IL10.
10 9. The method of Claim 8, wherein said anti-ELl 0 antibody is a
humanized or human monoclonal antibody.
10. The method of Claim 9, wherein said anti-lLl 0 antibody is
administered at a dosage of 0.01 to 1000 mg/kg body weight.
15
1 1 . The method of Claim 1 0, wherein the dosage of antibody ranges from
about 0. 1 to 50 mg/kg of body weight.
12. The method of Claim 1, wherein said anti-cytokine antibody is
20 administered concurrently with and/or prior to said chemotherapeutic agent.
13. The method of Claim 12, wherein said anti-cytokine antibody is
administered concurrently or from about one hour to thirty days prior to
administration of the chemotherapeutic agent.
25
14. The method of Claim 1, wherein the serum of said lymphoma patient is
tested for cytokine profiles prior to administration of said anti-cytokine antibody or
fragment thereof or antagonist.
-59-
WO 01/74388
PCT/US01/10382
15. A kit for administering the antibody or antagonist according to the
method of Claim 1.
16. A kit for testing cytokine profile according to the method of Claim 14.
5
17. A kit for testing cytokine profile and administering antibody or
antagonist according to the method of Claim 16.
18. A method of avoiding, decreasing or overcoming the resistance of
10 hematologic malignant cells to a therapeutic agent, comprising administering an anti-
cytokine antibody or cytokine antagonist to a patient diagnosed with a hematologic
malignancy.
19. A method of avoiding, decreasing or overcoming the resistance of
1 5 hematologic malignant cells to apoptosis induced by a therapeutic agent, comprising
administering an anti-cytokine antibody or cytokine antagonist to a patient diagnosed
with a hematologic malignancy.
20. The method of Claim 1 8, wherein said malignancy is a B cell
20 lymphoma or leukemia.
21. The method of Claim 19, wherein said malignancy is a B cell
lymphoma or leukemia.
25 22. A method of treating a patient with a hematologic malignancy who has
relapsed following chemotherapy, comprising administering an anti-cytokine antibody
or fragment thereof or cytokine antagonist to said patient.
-60-
WO 01/74388 PCT/US01/10382
23. A method of treating a patient having a hematologic malignancy who
is refractory to chemotherapy, comprising adrriinistering an anti-cytokine antibody or
fragment thereof or cytokine antagonist to said patient.
5 24. The method of Claim 21 , wherein said malignancy is a B cell
lymphoma or leukemia.
25 . A method of treating a patient with a hematologic malignancy who has
relapsed following therapy with a therapeutic antibody or fragment, comprising
10 administering an anti-cytokine antibody or fragment or cytokine antagonist to said
patient.
26. The method of Claim 23, wherein said therapeutic antibody is an anti-
CD20, anti-CD 19, anti-CD22, anti-CD37, anti-CD40, or anti-CD28 antibody.
15
27 . The method of treating a patient with a hematologic malignancy who is
refractory to therapy with a therapeutic antibody, comprising administering an anti-
cytokine antibody or fragment or cytokine antagonist to said patient.
20 28 . The method of Claim 25 , wherein said hematologic malignancy is B
cell lymphoma or leukemia.
29. A method of treating a B cell lymphoma patient comprising
administering to said patient a therapeutically effective amount of a B cell depleting
25 antibody simultaneously with or consecutively with in either order an anti-cytokine
antibody or fragment.
30. The method of Claim 29 wherein such B cell depleting antibody binds
a B cell antigen selected from the group consisting of CD19, CD20, CD22, CD23,
-61 -
WO 01/74388
PCT/US01/10382
CD27, CD37, CD53, CD72, CD73, CD74, CD©78, CD79a, CD79b, CD80, CD81,
CD82, CD83, CDw84, CD85 and CD86.
3 1 . The method of Claim 2 1 wherein said B cell depleting antibody binds
5 CD20.
32. The method of Claim 29 wherein said B cell depleting antibody binds
CD22.
10 33. The method of Claim 29, further comprising adrninistration of at least
one chemotherapeutic agent.
34. The method of Claim 33, wherein said at least one chemotherapeutic
agent is selected from the group consisting of CHOP, ICE, Mitozantrone, Cytarabine,
1 5 DVP, ATRA, Idarubicin, hoelzer chemotherapy regime, La La chemotherapy regime,
ABVD, CEOP, 2-CdA, FLAG & IDA with or without subsequent G-CSF treatment),
VAD, M & P, C- Weekly, ABCM, MOPP, DHAP, daunorubicin, doxorubicin,
methotrexate, and cisplatin.
20 35. The method of Claim 33, wherein said anti-cytokine antibody or
antagonist is administered prior to said anti-CD20 antibody and said at least one
chemotherapeutic agent.
36. The method of Claim 29, wherein said anti-cytokine antibody or
25 antagonist is administered prior to said anti-CD20 antibody.
37. The method of Claim 29, wherein said cytokine is selected from the
group consisting of EL2, IL6, IL10 and TNF-alpha.
30 38. The method of Claim 3 7, wherein said cytokine is IL10.
-62-
WO 01/74388
PCT/US01/10382
39. The method of Claim 29, wherein said anti-CD20 antibody is a
chimeric, humanized or human anti-CD20 antibody.
5 40. The method Claim of 39, wherein said anti-CD20 antibody is a
chimeric anti-CD20 antibody.
41 . The method of Claim 40, where said chimeric anti-CD20 antibody is
Rituximab®.
10
42. The method of Claim 41, wherein said Rituximab® is administered at
a dosage of 0.4 to 20 mg/kg body weight.
43. The method of Claim 33, wherein said at least one chemotherapeutic
1 5 agent is part of a CHOP chemotherapeutic regimen.
44. The method of Claim 29, wherein said B cell lymphoma is selected
from the group consisting of low grade/ follicular non-Hodgkin's lymphoma (NHL),
small lymphocytic (SL) NHL, intermediate grade/ follicular NHL, intermediate grade
20 diffuse NHL, high grade immunoblastic NHL, high grade lymphoblastic NHL, high
grade small non-cleaved cell NHL, bulky disease NHL and Waldenstrom's
Macroglobulinemia.
45. The method of Claim 44, wherein said B cell lymphoma is non-
25 Hodgkin's lymphoma (NHL).
46. The method of Claim 45, wherein said B cell lymphoma is low-grade,
follicular NHL.
-63-
WO 01/74388 PCT/US01/10382
47. The method of Claim 29, wherein the serum of said lymphoma patient
is tested for cytokine profiles prior to administration of said anti-cytokine antibody or
antagonist.
5 48. A kit for administering the anti-CD20 antibody and the anti-cytokine
antibody or antagonist according to the method of Claim 29.
49. A kit for testing cytokine profile and adrninistering anti-CD20 antibody
and anti-cytokine antibody or antagonist according to the method of Claim 42.
10
50. A method for treating a tumor having B cell involvement comprising
administering to a patient in need of such treatment an effective amount of an
antibody specific to a cytokine and a B cell depleting antibody which binds to an
antigen expressed by B cells.
15
51. A kit for administering the anti-CD20 antibody and the anti-cytokine
antibody or antagonist according to the method of Claim 50.
52. The method of Claim 50 wherein said anti-cytokine antibody binds to a
20 cytokine selected from the group consisting of an interferon, interleukin, tumor
necrosis factor, and colony stimulating factor.
53 . The method of Claim 5 1 wherein said anti-cytokine antibody binds to a
cytokine selected from the group consisting of an interferon, interleukin, tumor
25 necrosis factor, and colony stimulating factor.
54. The method of Claim 50 wherein said anti-cytokine antibody
specifically binds IL-10.
-64-
WO 01/74388
PCT/US01/10382
55. The method of Claim 5 1 wherein said anti-cytokine antibody
specifically binds IL- 10.
56. The method of Claim 50 wherein the B cell antigen is selected from the
5 group consisting of CD19, CD20, CD22, CD23, CD27, CD37, CD53, CD72, CD73,
CD74, CD©78, CD79a, CD79b, CD80, CD81, CD82, CD83, CDw84, CD85 and
CD86.
57. A kit for administering the anti-CD20 antibody and the anti-cytokine
10 antibody or antagonist according to the method of Claim 51.
58. The method of Claim 56 wherein said B cell antigen is CD20.
59. The method of Claim 58 wherein the anti-CD20 antibody is a human,
15 humanized or chimeric anti-CD20 antibody.
60. The method of Claim 59 wherein said antibody possesses ADCC
and/or CDC activity.
20 61 . The method of Claim 59 wherein said anti-CD20 induces apoptosis of
B cells.
62. The method of Claim 59 wherein said anti-CD20 antibody is
Rituxan®, a chimeric anti-CD20 antibody produced by ATCC 691 19.
25
63 . The method of Claim 5 1 wherein said patient comprises a solid non-
lymphoid tumor associated with a cancer selected from the group consisting of liver
cancer, head and neck cancer, breast cancer, prostate cancer, testicular cancer, ovarian
cancer, lung cancer, esophageal cancer, tracheal cancer, kidney cancer, bladder cancer,
30 and colorectal cancer.
-65-
WO 01/74388
PCT/US01/10382
64. The method of Claim 50 wherein said B cell lymphoma is selected
from the group consisting of low grade/ follicular non-Hodgkin's lymphoma (NHL),
small lymphocytic (SL) NHL, intermediate grade/ follicular NHL, intermediate grade
diffuse NHL, high grade immunoblastic NHL, high grade lymphoblastic NHL, high
5 grade small non-cleaved cell NHL, bulky disease NHL and Waldenstrom' s
Macroglobulinemia.
65. The method of Claim 50 wherein said antibodies are administered by
intravenous, intramuscular, intratumoral or intraperitoneal administration.
10
66. The method of Claim 51 wherein said antibodies are administered by
intravenous, intramuscular, intratumoral or intraperitoneal administration.
67. The method of Claim 5 1 when said solid tumor comprises a precancer,
15 early stage (Stage I or II solid cancer), advanced cancer (after Stage II cancer) or
metastasized cancer.
68 . The method of Claim 5 1 wherein said patient has colorectal cancer or
lung cancer.
20
69. A method of treating colorectal cancer or lung cancer having B cell
involvement comprising administering to a patient in need of such treatment an
effective amount of an antibody specific to DL-1 0 and a depleting anti-CD20 antibody.
25 70. The method of Claim 69 wherein said depleting anti-CD20 antibody is
a human, humanized or chimeric antibody.
7 1 . The method of Claim 70 wherein said antibody is Rituxan® produced
by ATCC 69119.
30
-66-
WO 01/74388
PCT/US01/10382
72. A method of treating B cell lymphoma in a patient in need of such
treatment which treatment includes the administration of an anti-ILlO antibody.
73 . A method of treating non-Hodgkin' s lymphoma in a patient in need of
5 such treatment which method comprises the administration of at least one anti-ILl 0
antibody.
74. A method of treating B cell lymphoma in a patient in need of such
treatment which method includes the administration of anti-ILl 0 antibody and at least
10 one B cell depleting antibody.
75 . The method of Claim 63 wherein said B cell depleting antibody binds
to a B cell antigen from the group consisting of CD 19, CD20, CD22, CD23, CD27,
CD37, CD53, CD72, CD73, CD74, CDco78, CD79a, CD79b, CD80, CD81, CD82,
15 CD83, CDw84, CD85 and CD86.
76. A method of treating B cell lymphoma in a patient in need of such
treatment which method comprises the administration of an anti-ILl 0 antibody and a
B cell depleting anti-CD20 or anti-CD22 antibody.
20
77. A method of treating B cell lymphoma in a patient in need of such
treatment comprising the administration of an anti-ILl 0 antibody and a B cell
depleting anti-CD20 antibody.
25 78 . A method of treating non-Hodgkin' s lymphoma in a patient in need of
such treatment comprising the administration of an anti-ILl 0 antibody and a B cell
depleting antibody.
-67-
WO 01/74388
PCT/US01/10382
79. A method of treating non-Hodgkin' s lymphoma in a patient in need of
such treatment comprising the administration of an anti-ILl 0 antibody and a B cell
depleting anti-CD20 antibody.
5 80. A method of treating non-Hodgkin's lymphoma in a patient in need of
such treatment comprising the aciministration of an anti-ILl 0 antibody and a B cell
depleting anti-CD22 antibody.
8 1 . The method of Claim 77 wherein said antibody is Rituxan® .
10
82. The method of Claim 79 wherein said antibody is Rituxan®.
83 . A combination therapy for treating B cell lymphoma in a patient
comprising the administration of a therapeutically effective amount of an anti-ILl 0
15 antibody, a B cell depleting anti-CD20 antibody and chemotherapy.
84. The method of Claim 83 wherein said anti CD20 antibody is
Rituxan®.
20 85. The method of Claim 83 wherein said patient has relapsed following
previous treatment with a B cell depleting antibody.
86. The method of Claim 85 wherein said antibody is Rituxan®.
-68-
INTERNATIONAL SEARCH REPORT
Interr si Application No
PCT/US 01/10382
A. CLASSIFICATION OF SUBJECT MATTER , , , , , ,
IPC 7 A61K39/395 A61P35/00 //C07K16/24,C07K16/28,(A61K39/395,
31:00)
According lo Inlernalional Patent Classification (IPC) or lo both national classification and IPC
B. FIELDS SEARCHED ■
Minimum documentation searched (classification system followed by classification symbols)
IPC 7 C07K A61K
Documentation searched other than minimum documentation to the extent that such documents are included in the fields searched
Electronic data base consulted during the international search (name of data base and, where practical, search terms used)
EPO-Internal , WPI Data, PAJ, BIOSIS
C. DOCUMENTS CONSIDERED TO BE RELEVANT
Category ° Citation of document, with indication, where appropriate, of the relevant passages
Relevant to claim No.
DE 295 12 719 U (MARX UWE DR MED ; SABAT
ROBERT (DE); GLASER RALF DR RER NAT (DE);
S) 29 August 1996 (1996-08-29)
page 2, paragraph 1
page 3, paragraph 5
72,73
1-14,
18-47,
50,
52-56,
58-71,
74-86
-/»
Further documents are listed in the continuation of box C.
Patent family members are listed In annex.
° Special categories of cited documents :
'A' document defining the general state of the art which is no)
considered to be of particular relevance
'E' earlier document but published on or after the international
filing date
"L" document which may throw doubts on priority clalm(s) or
which is cited to establish the publication date of another
citation or other special reason (as specified)
"O" document referring to an oral disclosure, use, exhibition or
other means
"P* document published prior lo the International filing date but
later than the priority date claimed
T later document published after the international filing date
or priority date and not in conflict with the application but
cited to understand the principle or theory underlying the
Invention
'X' document of particular relevance; the claimed Invention
cannot be considered novel or cannot be considered to
involve an inventive step when the document is taken alone
"Y" document of particular relevance; the claimed Invention
cannot be considered to involve an Inventive step when the
document is combined with one or more other such docu-
ments, such combination being obvious to a person skilled
In the art.
'&' document member of the same patent family
Date of the actual completion of the International search
28 August 2001
Date of mailing of the international search report
10/09/2001
Name and mailing address of the ISA
European Patent Office, P.B. 5618 Patentlaan 2
NL-2280HVRijswtjk
Tel (+31-70) 340-2040, TX. 31 651 epo nl,
Fax (+31-70)340-3016
Authorized officer
Muller-Ihomalla, K
Foim PC171SA/210 (second sheat) (July 1892)
page 1 of 3
INTERNATIONAL SEARCH REPORT
Intern 1 Application No
PCT/US 01/10382
^Continuation) DOCUMENTS CONSIDERED TO BE RELEVANT
Category"
Citation of document, with indlcation,where appropriate, of the relevant passages
Relevant to claim No.
X
WO 94 04180 A (SCHERING CORP)
72,73
3 March 1994 (1994-03-03)
abstract
page 1, line 9 -page 2, line 9
Y
claims 1-11
1-14,
18-47,
50,
52-56,
58-71 ,
74-86'
Y
CZUCZMAN M S ET AL: "Treatment of
1-14,
patients with low-grade B-cell Lymphoma
18-47,
with the combination of chimeric anti-CD20
50,
monoclonal antibody and CHOP chemotherapy"
52-56,
JOURNAL OF CLINICAL ONCOLOGY,
58-71,
PHILADELPHIA, PA, US,
74-86
vol. 17, no. 1, January 1999 (1999-01),
pages 268-276, XP000952705
abstract
page 268, column 1, line 1 -page 269,
column 2, paragraph 2
page 270, column 1, paragraph 3 -column 2,
paragraph 3
page 275, column 2; paragraph 2
Y
DATABASE BI0SIS 'Online'
LS (1 1 f \ \J fl W L_> W. A W w X w Will 1 1 1 v •
29-47,
BIOSCIENCES INFORMATION SERVICE
50
PHILADELPHIA PA US* 1997
52-56,
DEMIDEM AICHA ET AL- "Chimeric ant1-CD20
58-71^
(IDEC-C2B8) monoclonal antlbodv sensitizes
74-86'
a B cell lymphoma cell line to cell
killing by cytotoxic drugs."
Database accession no. PREV199799709997
XP002176012
abstract
& CANCER BIOTHERAPY &
RADIOPHARMACEUTICALS,
vol. 12, no. 3, 1997, pages 177-186,
ISSN: 1084-9785
Y
BUSKE C ET AL: "Monoclonal antibody
29-47, I
therapy for b cell non-Hodgkin's
50,
lymphomas: emerging concepts of a
52-56,
tumour-targeted strategy"
58-71,
EUROPEAN JOURNAL OF CANCER, PERGAM0N
74-86
PRESS, OXFORD, GB,
vol. 35, no. 4, 1999, pages 549-557,
XP001010018
ISSN: 0959-8049
page 550, column 1, paragraph 4 -page 553,
column 2, paragraph 1
page 555, column 1, last paragraph
_/-
Foim PCT/1SA/210 (continuation of second shaot) (July 1992)
page 2 of 3
INTERNATIONAL SEARCH REPORT lnterr , 1AppllcatlonNo
PCT/US 01/10382
C.(Contlnuatlon) DOCUMENTS CONSIDERED TO BE RELEVANT
Category •
Citation of document, with Indlcation.where appropriate, of the relevant passages
Relevant to claim No.
A
CORTES J KURZROCK R: "Inter! eukin-10 in
non-Hodgk1n's lymphoma"
LEUKEMIA AND LYMPHOMA, HARWOOD ACADEMIC
PUBLISHERS, CHUR, CH,
vol. 26, no. 3/4, July 1997 (1997-07),
pages 251-259, XP001020774
ISSN: 1042-8194
the whole document
1-14,
18-47,
50,
52-56,
58-86
Form FCT/1SA/210 (continuation of second shoot) (July 1892)
page 3 of 3
INTERNATIONAL SEARCH REPORT
International Application No. PCTAlS 01 A0382
FURTHER INFORMATION CONTINUED FROM PCT/ISA/ 210
Continuation of Box 1.2
Claims Nos.: 15-17,48,49,51,57
The kits according to claims 15-17,48,49,51,57 have not been searched, as
the subject-matter of said claims 1s not defined 1n that the latter lack
concrete technical features defining the kit 1n questions. An attempt is
made to define said kits by reference to a result to be achieved. Again,
this lack of clarity 1n the present case is such as to render a
meaningful search over the whole of the claimed scope impossible.
The applicant's attention is drawn to the fact that claims, or parts of
claims, relating to Inventions in respect of which no international
search report has been established need not be the subject of an
international preliminary examination (Rule 66.1(e) PCT). The applicant
is advised that the EPO policy when acting as an International
Preliminary Examining Authority is normally not to carry out a
preliminary examination on matter which has not been searched. This is
the case Irrespective of whether or not the claims are amended following
receipt of the search report or during any Chapter II procedure.
INTERNATIONAL SEARCH REPORT
Information on patent family members
Inten u Application No
PCT/US 01/10382
Patent document
cited in search report
Publication
date
Patent family
member(s)
Publication
date
DE 29512719 U 29-08-1996 NONE
WO 9404180 A 03-03-1994
IIC
Uo
COCQOC/t
0000004
A
Oft 11 IQOil
zy— n-iyy4
AT
Ml
1 /\9Q/I7
lo^y^/
T
1
lb- uz-iyys
Al 1
AU
O8O0Z8
n
D
A7 no 1 ftft"7
0/ -08-199/
Al 1
AU
cai nono
5010893
A
1 c no i ftftii
ib-03-1994
r a
LA
Ol A OOC1
Z14Z861
A
no no inftji
03-03-1994
ioyozo4
A
no no inftii
03-08-1994
U
yb00441
A
A
in 1 n 1 ftftC
18-10-1995
Ut
oyoioyzi
n
V
1Z-03-1998
ut
6y3it>9Zi
T
1
f\A nc infto
04-06-1998
iw
UK
o/i933
T
1
oo nn iaao
£3-09-1998
CD
tr
ft£"71 noo
00/1933
A
A
Oft ftft IftftC
ZO-09-1995
CD
tr
nom o.c i
Uoiuybi
A
A
oo in i nm
ZZ-10-1997
CO
to
Ol 1 1 7£rt
ziii/69
T
1
i£ no i ftfto
16-03-1998
CT
r 1
youoy/
A
A
A7 ft A 1AAC
0/-U4-iyyb
TD
30ZO&35
T
1
oi n*7 mfto
31-07-1998
HK.
10043ZO
A
A
Oft 11 1 nno
ZU-1 1-1998
lji i
HU
/oy/o
A
A
OO 11 1 ftftC
Z8-1 1-1995
T 1
1L
100/Zb
A
A
n£ i o onnn
06-12-2000
ID
JP
8500362
T
16-01-1996
KR
170037
D
D
m no i nnn
01-02-1999
MX
9305054
A
A
oo no 1 nn a
Z8-02-1994
NO
950600
A
20-04-1995
NZ
255757
A
27-07-1997
NZ
314940
A
29-07-1999
PL
307566
A
29-05-1995
RU
2120802
C
27-10-1998
SG
52248
A
28-09-1998
SK
21195
A
01-10-1996
ZA
9306060
A
23-05-1994
US
6106823
A
22-08-2000
US
5601815
A
11-02-1997
Form PCTrtSA/210 (patera lemily annex) (July 18S2)
Live Music Archive
Librivox Free Audio
Metropolitan Museum
Cleveland Museum of Art
Internet Arcade
Console Living Room
Books to Borrow
Open Library
TV News
Understanding 9/11